Quinolinone-carboxamide compounds as 5-HT4 receptor agonists

ABSTRACT

The invention provides novel quinolinone-carboxamide 5-HT4 receptor agonist compounds of Formula (I). The invention also provides pharmaceutical compositions comprising such compounds, the use such compounds to treat diseases associated with 5-HT4 receptor activity, and processes and intermediates useful for preparing such compounds. Wherein; R 1  is hydrogen, halo, hadroxy, C 1-4  alkyl, or C 1-4  alkoxy; R 2  is C 3-4  alkyl, or C 3-6 cycloakyl; R 3  is hydrogen or C 1-3  alkyl: R 4  is —S(O) 2  R 6  or —C(O)R 7 ; R 5  is hydrogen, C 1-3 alkyl, C 2-3  alkyl substituted with —OH or C 1-3  alkoxy, or —CH 2 -pyrydyl; R 6  is C 1-3  alkyl; or R 5  and R 6  taken together from C 3-4  alkylenyl; and R 7  is hydrogen, C 1-3 alkyl, or pyrydyl; or pharmaceutically-acceptable salt or solvate or stereoisomer thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/560,076, filed on Apr. 7, 2004, the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to quinolinone-carboxamide compounds which areuseful as 5-HT₄ receptor agonists. The invention is also directed topharmaceutical compositions comprising such compounds, methods of usingsuch compounds for treating or preventing medical conditions mediated by5-HT₄ receptor activity, and processes and intermediates useful forpreparing such compounds.

2. State of the Art

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter that iswidely distributed throughout the body, both in the central nervoussystem and in peripheral systems. At least seven subtypes of serotoninreceptors have been identified and the interaction of serotonin withthese different receptors is linked to a wide variety of physiologicalfunctions. There has been, therefore, substantial interest in developingtherapeutic agents that target specific 5-HT receptor subtypes.

In particular, characterization of 5-HT₄ receptors and identification ofpharmaceutical agents that interact with them has been the focus ofsignificant recent activity. (See, for example, the review by Langloisand Fischmeister, J. Med. Chem. 2003, 46, 319-344.) 5-HT₄ receptoragonists are useful for the treatment of disorders of reduced motilityof the gastrointestinal tract. Such disorders include irritable bowelsyndrome (IBS), chronic constipation, functional dyspepsia, delayedgastric emptying, gastroesophageal reflux disease (GERD), gastroparesis,post-operative ileus, intestinal pseudo-obstruction, and drug-induceddelayed transit. In addition, it has been suggested that some 5-HT₄receptor agonist compounds may be used in the treatment of centralnervous system disorders including cognitive disorders, behavioraldisorders, mood disorders, and disorders of control of autonomicfunction.

Despite the broad utility of pharmaceutical agents modulating 5-HT₄receptor activity, few 5-HT₄ receptor agonist compounds are in clinicaluse at present. One agent, cisapride, that was utilized extensively fortreatment of motility disorders of the gastrointestinal tract waswithdrawn from the market, reportedly due to cardiac side effects. Latestage clinical trials of another agent, prucalopride, have beensuspended.

Accordingly, there is a need for new 5-HT₄ receptor agonists thatachieve their desired effects with minimal side effects. Preferredagents may possess, among other properties, improved selectivity,potency, pharmacokinetic properties, and/or duration of action.

SUMMARY OF THE INVENTION

The invention provides novel compounds that possess 5-HT₄ receptoragonist activity. Among other properties, compounds of the inventionhave been found to be potent and selective 5-HT₄ receptor agonists. Inaddition, compounds of the invention have been found to exhibitfavorable pharmacokinetic properties which are predictive of goodbioavailability upon oral administration.

Accordingly, the invention provides a compound of formula (I):

wherein:

R¹ is hydrogen, halo, hydroxy, C₁₋₄alkyl, or C₁₋₄alkoxy;

R² is C₃₋₄alkyl, or C₃₋₆cycloalkyl;

R³ is hydrogen or C₁₋₃alkyl;

R⁴ is —S(O)₂R⁶ or —C(O)R⁷;

R⁵ is hydrogen, C₁₋₃alkyl, C₂₋₃alkyl substituted with —OH or C₁₋₃alkoxy,or —CH₂-pyridyl;

R⁶ is C₁₋₃alkyl;

or, R⁵ and R⁶ taken together form C₃₋₄alkylenyl; and

R⁷ is hydrogen, C₁₋₃alkyl, or pyridyl;

or a pharmaceutically-acceptable salt or solvate or stereoisomerthereof.

The invention also provides a pharmaceutical composition comprising acompound of the invention and a pharmaceutically-acceptable carrier.

The invention also provides a method of treating a disease or conditionassociated with 5-HT₄ receptor activity, e.g. a disorder of reducedmotility of the gastrointestinal tract, the method comprisingadministering to the mammal, a therapeutically effective amount of acompound of the invention.

Further, the invention provides a method of treating a disease orcondition associated with 5-HT₄ receptor activity in a mammal, themethod comprising administering to the mammal, a therapeuticallyeffective amount of a pharmaceutical composition of the invention.

The compounds of the invention can also be used as research tools, i.e.to study biological systems or samples, or for studying the activity ofother chemical compounds. Accordingly, in another of its method aspects,the invention provides a method of using a compound of formula (I), or apharmaceutically acceptable salt or solvate or stereoisomer thereof, asa research tool for studying a biological system or sample or fordiscovering new 5-HT₄ receptor agonists, the method comprisingcontacting a biological system or sample with a compound of theinvention and determining the effects caused by the compound on thebiological system or sample.

In separate and distinct aspects, the invention also provides syntheticprocesses and intermediates described herein, which are useful forpreparing compounds of the invention.

The invention also provides a compound of the invention as describedherein for use in medical therapy, as well as the use of a compound ofthe invention in the manufacture of a formulation or medicament fortreating a disease or condition associated with 5-HT₄ receptor activity,e.g. a disorder of reduced motility of the gastrointestinal tract, in amammal.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides novel quinolinone-carboxamide 5-HT₄ receptoragonists of formula (I), or pharmaceutically-acceptable salts orsolvates or stereoisomers thereof. The following substituents and valuesare intended to provide representative examples of various aspects ofthis invention. These representative values are intended to furtherdefine such aspects and are not intended to exclude other values orlimit the scope of the invention.

In a specific aspect of the invention, R¹ is hydrogen, halo, C₁₋₄alkyl,or C₁₋₄alkoxy.

In other specific aspects, R¹ is hydrogen, halo, or C₁₋₄alkyl; or R¹ ishydrogen or halo; or R¹ is fluoro; or R¹ is bromo.

In yet another specific aspect, R¹ is hydrogen.

In a specific aspect, R² is C₃₋₄alkyl or C₃₋₆cycloalkyl.

In another specific aspect, R² is C₃₋₄alkyl. Representative R² groupsinclude n-propyl, isopropyl, n-butyl, sec-butyl, and tert-butyl.

In another specific aspect, R² is isopropyl.

In yet other specific aspects R² is C₃₋₄alkyl or C₄₋₅cycloalkyl; or R²is isopropyl or C₄₋₅cycloalkyl.

In a specific aspect, R³ is hydrogen or C₁₋₃alkyl.

In other specific aspects, R³ is hydrogen, or R³ is methyl.

In a specific aspect, R⁴ is —S(O)₂R⁶ wherein R⁶ is C₁₋₃alkyl.

In another specific aspect, R⁴ is —S(O)₂CH₃.

In a specific aspect, R⁴ is —C(O)R⁷ wherein R⁷ is hydrogen, C₁₋₃alkyl orpyridyl.

In other specific aspects, R⁴ is —C(O)R⁷ wherein R⁷ is hydrogen orC₁₋₃alkyl; or R⁴ is —C(O)R⁷ wherein R⁷ is hydrogen or methyl; or R⁴ is—C(O)R⁷ wherein R⁷ is hydrogen; or R⁴ is —C(O)R⁷ wherein R⁷ is methyl.

In yet another specific aspect, R⁴ is —C(O)R⁷ wherein R⁷ is 3-pyridyl or4-pyridyl.

In a specific aspect, R⁵ is hydrogen; C₁₋₃alkyl; C₂₋₃alkyl substitutedwith —OH or C₁₋₃alkoxy; or —CH₂-pyridyl.

In other specific aspects R⁵ is hydrogen, C₁₋₃alkyl, or —CH₂-pyridyl; orR⁵ is hydrogen or C₁₋₃alkyl.

In yet other specific aspects, R⁵ is —CH₂-3-pyridyl; or R⁵ is hydrogenor methyl; or R⁵ is hydrogen; or R⁵ is methyl.

In yet other specific aspects, R⁵ and R⁶ taken together form —(CH₂)₃— or—(CH₂)₄; or R⁵ and R⁶ taken together form —(CH₂)₃—.

In one aspect, the invention provides a compound of formula (I) whereinR³ is hydrogen.

In another aspect, the invention provides a compound of formula (I)wherein R⁴ is —S(O)₂R⁶.

In another aspect, the invention provides a compound of formula (I)wherein R⁴ is —C(O)R⁷

The invention further provides a compound of formula (I) wherein R¹ ishydrogen or halo; R² is isopropyl or C₄₋₅cycloalkyl; and R³, R⁴, R⁵, R⁶,and R⁷ are defined as in formula (I).

In yet another aspect, the invention provides a compound of formula (I)wherein:

R¹ is hydrogen;

R² is C₃₋₄alkyl or C₄₋₅cycloalkyl;

R³ is hydrogen;

R⁴ is —S(O)₂R⁶ or —C(O)R⁷;

R⁵ is hydrogen or C₁₋₃alkyl;

R⁶ is C₁₋₃alkyl; and

R⁷ is hydrogen or C₁₋₃alkyl.

In yet another aspect, the invention provides a group of compounds offormula (II):

wherein R¹ is hydrogen, R² is isopropyl, and R³, R⁴, R⁵, and R⁶, or R³,R⁴, R⁵, and R⁷ take the values shown in Table I and Table II,respectively.

TABLE I R⁴ = —S(O)₂R⁶ Example No. R³ R⁵ R⁶ 1 H CH₃ CH₃ 2 CH₃ CH₃ CH₃ 3CH₃ —CH₂-3-pyridyl CH₃ 4 H H CH₃ 5 H —CH₂-3-pyridyl CH₃ 6 H CH₃ CH₃ 7 HCH₃ CH₃ 21 H C₂H₅ CH₃ 22 H H CH₃ 23 H —(CH₂)₃—

TABLE II R⁴ = —C(O)R⁷ Example No. R³ R⁵ R⁷ 8 H CH₃ 4-pyridyl 9 H H4-pyridyl 10 CH₃ CH₃ CH₃ 11 CH₃ CH₃ 4-pyridyl 12 CH₃ —CH₂-3-pyridyl CH₃13 H H CH₃ 14 H CH₃ CH₃ 15 H CH₃ H 16 H H H 17 H CH₃ CH₃ 18 H CH₃ H

The chemical naming conventions used herein are illustrated for thecompound of Example 1:

which is designated 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylicacid{(1S,3R,5R)-8-[2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amide,according to the AutoNom software, provided by MDL Information Systems,GmbH (Frankfurt, Germany). The designation (1S,3R,5R) describes therelative orientation of the bonds associated with the bicyclic ringsystem that are depicted as solid and dashed wedges. The compound isalternatively denoted asN-[(3-endo)-8-[2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl]-1-(1-methylethyl)-2-oxo-1,2-dihydro-3-quinolinecarboxamide.In all of the compounds of the invention depicted above, thequinolinone-carboxamide is endo to the azabicyclooctane group.

Particular mention may be made of the following compounds1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;

1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[2-hydroxy-3-(methanesulfonylamino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;

1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;

1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(S)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;

1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-(acetyl-methyl-amino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;

1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-(formyl-methyl-amino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;

1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-3-(acetyl-methyl-amino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;

1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-3-(formyl-methyl-amino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;and

1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonylamino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide.

As exemplified by particular compounds listed above, the compounds ofthe invention may contain a chiral center, specifically, at the carbonatom in formulas (I) or (II) bearing the substituent —OR³. Accordingly,the invention includes racemic mixtures, pure stereoisomers, andstereoisomer-enriched mixtures of such isomers, unless otherwiseindicated. When a particular stereoisomer is shown, it will beunderstood by those skilled in the art, that minor amounts of otherstereoisomers may be present in the compositions of the invention unlessotherwise indicated, provided that any utility of the composition as awhole is not eliminated by the presence of such other isomers.

Definitions

When describing the compounds, compositions and methods of theinvention, the following terms have the following meanings, unlessotherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched or combinations thereof. Unless otherwisedefined, such alkyl groups typically contain from 1 to 10 carbon atoms.Representative alkyl groups include, by way of example, methyl, ethyl,n-propyl (n-Pr), isopropyl (i-Pr), n-butyl (n-Bu), sec-butyl, isobutyl,tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl andthe like.

The term “alkylenyl” means a divalent saturated hydrocarbon group whichmay be linear or branched or combinations thereof. Unless otherwisedefined, such alkylenyl groups typically contain from 1 to 10 carbonatoms. Representative alkylenyl groups include, by way of example,methylene, ethylene, n-propylene, n-butylene, propane-1,2-diyl(1-methylethylene), 2-methylpropane-1,2-diyl (1,1-dimethylethylene) andthe like.

The term “alkoxy” means a monovalent group —O-alkyl, where alkyl isdefined as above. Representative alkoxy groups include, by way ofexample, methoxy, ethoxy, propoxy, butoxy, and the like.

The term “cycloalkyl” means a monovalent saturated carbocyclic groupwhich may be monocyclic or multicyclic. Unless otherwise defined, suchcycloalkyl groups typically contain from 3 to 10 carbon atoms.Representative cycloalkyl groups include, by way of example,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, and the like.

The term “halo” means fluoro, chloro, bromo or iodo.

The term “compound” means a compound that was synthetically prepared orprepared in any other way, such as by metabolism.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treatment” as used herein means the treatment of a disease,disorder, or medical condition in a patient, such as a mammal(particularly a human) which includes:

-   -   (a) preventing the disease, disorder, or medical condition from        occurring, i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease, disorder, or medical condition,        i.e., eliminating or causing regression of the disease,        disorder, or medical condition in a patient;    -   (c) suppressing the disease, disorder, or medical condition,        i.e., slowing or arresting the development of the disease,        disorder, or medical condition in a patient; or    -   (d) alleviating the symptoms of the disease, disorder, or        medical condition in a patient.

The term “pharmaceutically-acceptable salt” means a salt prepared froman acid or base which is acceptable for administration to a patient,such as a mammal. Such salts can be derived frompharmaceutically-acceptable inorganic or organic acids and frompharmaceutically-acceptable bases. Typically,pharmaceutically-acceptable salts of compounds of the present-inventionare prepared from acids.

Salts derived from pharmaceutically-acceptable acids include, but arenot limited to, acetic, adipic, benzenesulfonic, benzoic,camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic,hydrobromic, hydrochloric, lactic, maleic, malic, mandelic,methanesulfonic, mucic, nitric, pantothenic, phosphoric, succinic,sulfuric, tartaric, p-toluenesulfonic, xinafoic (1-hydroxy-2-naphthoicacid), naphthalene-1,5-disulfonic acid and the like.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, i.e. a compound of the invention or apharmaceutically-acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include by way of example, water, methanol, ethanol,isopropanol, acetic acid, and the like. When the solvent is water, thesolvate formed is a hydrate.

It will be appreciated that the term “or a pharmaceutically-acceptablesalt or solvate of stereoisomer thereof” is intended to include allpermutations of salts, solvates and stereoisomers, such as a solvate ofa pharmaceutically-acceptable salt of a stereoisomer of a compound offormula (I).

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino nitrogen. Representativeamino-protecting groups include, but are not limited to, formyl; acylgroups, for example alkanoyl groups, such as acetyl; alkoxycarbonylgroups, such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups,such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc);arylmethyl groups, such as benzyl (Bn), trityl (Tr), and1,1-di-(4′-methoxyphenyl)methyl; silyl groups, such as trimethylsilyl(TMS) and tert-butyldimethylsilyl (TBDMS); and the like.

General Synthetic Procedures

Compounds of the invention can be prepared from readily availablestarting materials using the following general methods and procedures.Although a particular aspect of the present invention is illustrated inthe schemes below, those skilled in the art will recognize that allaspects of the present invention can be prepared using the methodsdescribed herein or by using other methods, reagents and startingmaterials known to those skilled in the art. It will also be appreciatedthat where typical or preferred process conditions (i.e., reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. Optimum reaction conditions may vary with theparticular reactants or solvent used, but such conditions can bedetermined by one skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group, as well assuitable conditions for protection and deprotection, are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

In one method of synthesis, compounds of formula (I) are prepared asillustrated in Scheme A. (The substituents and variables shown in thefollowing schemes have the definitions provided above unless otherwiseindicated).

In Scheme A, L represents a leaving group such as chloro, bromo, iodo,or ethoxy, or the reagent L-R⁴is the carboxylic acid HO—C(O)R⁷, i.e. Lformally represents hydroxy.

Optimal reaction conditions for the reaction of Scheme A may varydepending on the chemical properties of the reagent L-R⁴, as is wellknown to those skilled in the art.

For example, when L is a halo leaving group, such as chloro, thereaction is typically conducted by contacting intermediate (III) withbetween about 1 and about 4 equivalents of a compound of formula L-R⁴ inan inert diluent, such as dichloromethane, in the presence of an excessof base, for example between about 3 and about 6 equivalents, of base,such as N,N-diisopropylethylamine or 1,8-diazabicyclo[5.4.0]undec-7-ene(DBU). Suitable inert diluents also include N,N-dimethylformamide,trichloromethane, 1,1,2,2-tetrachloroethane, tetrahydrofuran, and thelike. The reaction is typically conducted at a temperature in the rangeof about −100° C. to about 30° C. for about a quarter hour to about 2hours, or until the reaction is substantially complete. Exemplaryreagents L-R⁴ in which L is chloro include methanesulfonylchloride andacetylchloride.

When the reagent L-R⁴ is a carboxylic acid, Scheme A represents an amidecoupling reaction which is typically conducted by contactingintermediate (III) with between about 1 and about 4 equivalents of acompound of a carboxylic acid L-R⁴ in an inert diluent, for example,N,N-dimethylformamide, in the presence of a coupling agent such asbenzotriazol-1-yloxytripyrrolidino-phosphonium hexafluorophosphate(PyBop). The reaction is typically conducted at ambient temperature, forabout a quarter hour to about 2 hours, or until the reaction issubstantially complete. Suitable alternative coupling agents include 1,3dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and PyBop combinedwith 1-hydroxy-7-azabenzotriazole (HOAt).

The amide coupling of intermediate (III) with the carboxylic acid L-R⁴alternatively can be performed by converting L-R⁴ to an activated ester,such as an N-hydroxy succinimide (NHS) ester or a p-nitrophenyl ester,or an acid imidazole, which is then reacted with intermediate (III).

Alternatively, when the reagent L-R⁴ is a liquid, for example ethylformate, the reaction can be performed by dissolving (III) in a largeexcess of the reagent L-R⁴, and heating to a temperature of betweenabout 50° C. and about 100° C. for about 12 to about 24 hours.

The product of formula (I) is isolated and purified by conventionalprocedures. For example, the product can be concentrated to drynessunder reduced pressure, taken up in an aqueous weak acid solution andpurified by HPLC chromatography.

Alternatively, compounds of formula (I) can be prepared by N-alkylatinga compound of formula (I) in which R² is hydrogen, which can be preparedaccording to Scheme A. The N-alkylation reaction is typically conductedby contacting a compound of formula (I) in which R² is hydrogen withbetween about 1 and about 4 equivalents of a compound of the formulaL′-R² in which L′ is a leaving group such as iodo or bromo. Thisreaction is typically conducted in a polar aprotic solvent such asdimethylformamide in the presence of between about 2 and about 4equivalents of strong base, such as potassium tert-butoxide. Typically,the reaction is performed at a temperature of between about 60° C. andabout 100° C. for between about 6 and about 24 hours, or until thereaction is substantially complete.

In yet another alternative, compounds of formula (I) in which R¹ isother than hydrogen are prepared by conventional processes fromcompounds of formula (I) in which R¹ is hydrogen.

Intermediates of formula (III) are prepared from readily availablestarting materials. For example, when the carbon bearing the substituent—OR³ is not chiral, an intermediate of formula (III) is prepared by theprocedure illustrated in Scheme B.

where L′ independently represents a halo leaving group such as bromo,chloro, or iodo. A negatively-charged counterion is also presentassociated with the positively-charged intermediate (V) or (V′).

First, an intermediate of formula (IV) is reacted with an oxiranecompound, for example, 2-bromomethyloxirane (commonly, epibromohydrin)to form an azetidine salt of formula (V). This reaction is typicallyconducted by contacting (IV) with between about 2 and about 4equivalents of 2-bromomethyloxirane in a polar diluent, such as ethanol.The reaction is typically conducted at ambient temperature for betweenabout 24 and about 48 hours or until the reaction is substantiallycomplete.

It will be understood that in the process of Scheme B and in otherprocesses described below using intermediate (IV), intermediate (IV) canbe supplied in the form of the freebase or in a salt form, withappropriate adjusment of reaction conditions, as necessary, as known tothose skilled in the art.

An intermediate of formula (V′), in which R³ is C₁₋₃alkyl, can beprepared by contacting intermediate (V) with from slightly less than oneequivalent to about one equivalent of a compound of formula L′-R³, whereR³ is C₁₋₃alkyl, in an inert diluent in the presence of between about 1and about 3 equivalents of a strong base, such as potassiumtert-butoxide or sodium hydride. The reaction is typically conducted atambient temperature for between about a quarter hour to an hour, oruntil the reaction is substantially complete. Suitable inert diluentsinclude dichloromethane, trichloromethane, 1,1,2,2-tetrachloroethane,and the like.

Next, the azetidine intermediate (V) or (V′) is reacted with an amine ofthe formula H₂NR⁵ to provide the intermediate (III). Typically, theazetidine intermediate is dissolved in an inert diluent, such asethanol, and contacted with between about 1 and about 8 equivalents ofthe amine H₂NR⁵. For example, when the amine H₂NR⁵ is a volatilereagent, such as methylamine, preferably, between about 5 and about 7equivalents of the amine are used. The reaction is typically conductedat a temperature of between about 50° C. and about 100° C. for betweenabout 12 and about 24 hours or until the reaction is substantiallycomplete.

An intermediate of formula (III) in which R⁵ is hydrogen, can beprepared from the azetidine intermediate (V) or (V′) using ammoniumformate in place of ammonia, i.e. in place of the reagent H₂NR⁵indicated in Scheme B. Alternatively, to prepare intermediate (III)where R⁵ is hydrogen, the azetidine ring of (V) or (V′) can be opened byreaction with an azide, such as sodium azide, which is then followed bya reduction reaction to provide intermediate (III), or the ring can beopened by reaction with ammonium hydroxide.

As described in detail in Example 4a, when R³ and R⁵ are hydrogen andthe carbon bearing the substituent —OR³ is not chiral, an intermediateof formula (III) can be prepared by reacting intermediate (IV) with anoxiranylmethyl compound having a protected nitrogen atom and thendeprotecting. One useful reagent is2-oxiranylmethyl-isoindole-1,3-dione, commonly epoxypropylphthalimide,which is reacted with intermediate (IV) to form an intermediate in whicha phthalimidyl-substituted 2-hydroxy propyl group:

is joined to the nitrogen of the azabicylcooctane ring of formula (IV).The phthalimidyl group is then removed by refluxing in hydrazine to forman intermediate of formula (III) in which R³ and R⁵ are hydrogen.

An intermediate of formula (III) in which R³ and R⁵ are hydrogen canalso be prepared by reaction of the azetidine (V) with the anion ofphthalimide and subsequent treatment with hydrazine.

In an alternative method of synthesis, an intermediate of formula (III)in which R³ is hydrogen, can be prepared by reaction of intermediate(IV) with a protected intermediate (VI):

followed by a deprotection step. In formula (VI), P¹ is anamino-protecting group, L′ is a halo leaving group, and the asteriskdenotes a chiral center. The process utilizing an intermediate offormula (VI) is useful for preparing forms of intermediate (III) inwhich the stereochemistry at the center marked by the aserisk isspecifically (R) or (S) as well as for preparing non-chiral forms ofintermediate (III).

Typically, intermediate (IV) is contacted with between about 1 and about2 equivalents of intermediate (VI) in a polar diluent, such as methanol,in the presence of more than one equivalent of a base, such asN,N-diisopropylethylamine. The reaction is typically conducted at atemperature of between about 60° C. and about 100° C. for between about12 and about 24 hours, or until the reaction is substantially complete.The protecting group P¹ is removed by standard procedures to provide anintermediate of formula (III). A useful protecting group P¹ is Boc,which is typically removed by treatment with an acid, such astrifluoroacetic acid.

In yet another alternative process for the preparation of intermediate(III), intermediate (VI) can first be converted to a cyclized form(VII):

before reaction with intermediate (IV) to provide intermediate (III).Intermediate (VII) is typically prepared by dissolving intermediate (VI)in an inert diluent, for example, tetrahydrofuran, in the presence ofbase, for example sodium hydroxide. The reaction of (VII) with (IV) toprovide intermediate (III) is typically performed by contactingintermediate (IV) with between about 1 and about 4 equivalents ofintermediate (VII) in a polar diluent, such as methanol. The reaction istypically conducted at a temperature of between about 60° C. to about100° C. for between about 1 and about 4 hours, or until the reaction issubstantially complete. The protecting group P¹ is removed by standardprocedures to provide an intermediate of formula (III).

The protected intermediate (VI) can be prepared from an oxirane asillustrated in Scheme C for the particular example of forming aBoc-protected chiral intermediate (VI′) using a chiral oxirane. Thereaction is equally useful for the preparation of non-chiral compoundsof formula (VI).

As shown in Scheme C, a benzylamine 2 is contacted with at least oneequivalent of a chiral oxirane 1 in a non-polar diluent such as hexaneor toluene to form the 2-hydroxypropylamine 3. The reaction is typicallyconducted at room temperature for between about 12 and about 24 hours,or until the reaction is substantially complete. The intermediate 3 istypically reacted with a slight excess of di-tert-butyl dicarbonate(commonly (Boc)₂O), for example, about 1.1 equivalents, under a hydrogenatmosphere in the presence of a transition metal catalyst to provide theBoc protected intermediate (VI′). The reaction is typically conducted atambient temperature for between about 8 to about 24 hours.

A process for preparing intermediates of formula (IV) is shown in SchemeD.

The protected aminoazabicyclooctane, or commonly, aminotropane 5 isfirst reacted with the substituted quinolinone carboxylic acid (VIII).Typically, this reaction is conducted by first converting (VIII) to anacid chloride by contacting (VIII) with at least one equivalent,preferably between about 1 and about 2 equivalents of an activatingagent, such as thionyl chloride or oxalyl chloride in an aromaticdiluent, such as toluene, benzene, xylene, or the like. The reaction istypically conducted at a temperature ranging from about 80° C. to about120° C. for about 15 minutes to about 4 hours, or until the reaction issubstantially complete.

The acid chloride solution is typically added to a biphasic mixture ofabout 1 equivalent of the aminotropane 5 to form a protectedintermediate, which is extracted by standard procedures. The biphasicmixture of 5 is generally prepared by dissolving 5 in an aromaticdiluent, such as used above, and adding an aqueous solution containingan excess of base, such as sodium hydroxide or potassium hydroxide,preferably about 2 to 5 equivalents of base.

Alternatively, the amide coupling of intermediate 5 with the carboxylicacid (VIII) can be performed in the presence of a coupling agent such as1,3 dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), orbenzotriazol-1-yloxytripyrrolidino-phosphonium hexafluorophosphate(PyBop), optionally combined with 1-hydroxy-7-azabenzotriazole (HOAt),as described above for the amide coupling of intermediate (III) with acarboxylic acid. In yet another alternative, the amide coupling ofintermediate 5 with the carboxylic acid (VIII) can be performed byconverting (VIII) to an activated ester, also described above.

The protecting group P¹ is removed by standard procedures to provide anintermediate of formula (IV). For example when the protecting group isBoc, typically removal is by treatment with an acid, such astrifluoroacetic acid, providing the acid salt of the intermediate. Theacid salt of intermediate (IV) can be converted to the free base, ifdesired, by conventional treatment with base. The protecting group Cbz,for another example, is conveniently removed by hydrogenolysis over asuitable metal catalyst such as palladium on carbon.

The protected aminotropane 5 employed in the reactions described in thisapplication is prepared from readily available starting materials. Forexample, when the protecting group P¹ is Boc, the protected aminotropane5′ is prepared by the procedure illustrated in Scheme E.

As described in detail in Example 1a below, to prepare the protectedintermediate 5′, first, 2,5-dimethoxy tetrahydrofuran 6 is contactedwith between about 1 and 2 equivalents, preferably about 1.5 equivalentsof benzyl amine and a slight excess, for example about 1.1 equivalents,of 1,3-acetonedicarboxylic acid 7 in an acidic aqueous solution in thepresence of a buffering agent such as sodium hydrogen phosphate. Thereaction mixture is heated to between about 60 and about 100° C. toensure decarboxylation of any carboxylated intermediates in the product,8-benzyl-8-azabicyclo[3.2.1]octan-3-one 8, commonly N-benzyltropanone.

The intermediate 8 is typically reacted with a slight excess ofdi-tert-butyl dicarbonate (commonly (Boc)₂O), for example, about 1.1equivalents, under a hydrogen atmosphere in the presence of a transitionmetal catalyst to provide the Boc protected intermediate9,3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester.The reaction is typically conducted at ambient temperature for about 12to about 72 hours. Finally, intermediate 9 is contacted with a largeexcess, for example at least about 25 equivalents, of ammonium formatein an inert diluent, such as methanol, in the presence of a transitionmetal catalyst to provide the product 5′ in the endo configuration withhigh stereospecificity, for example endo to exo ratio of >99:1. Thereaction is typically conducted at ambient temperature for about 12 toabout 72 hours or until the reaction is substantially complete. It isadvantageous to add the ammonium formate reagent in portions. Forexample, intermediate 9 is contacted with an initial portion of ammoniumformate of about 15 to about 25 equivalents. After an interval of about12 to about 36 hours, an additional portion of about 5 to about 10equivalents of ammonium formate is added. The subsequent addition can berepeated after a similar interval. The product 5′ can be purified byconventional procedures, such as alkaline extraction.

In an alternative method of synthesis, compounds of formula (I) areprepared by coupling the substituted quinolinone carboxylic acid (VIII)with an intermediate of formula (IX) as illustrated in Scheme F.

The reaction of Scheme F is typically conducted under the amide couplingconditions described above for the reaction of the carboxylic acid(VIII) with intermediate 5.

Intermediates of formula (IX) can be prepared by deprotecting anintermediate of formula (X):

where P² represents an amino-protecting group.

Intermediates of formula (X) can be prepared from readily availablestarting materials using procedures analogous to the alkylation andother reactions described above and/or using alternative reactions wellknown to those skilled in the art. For example, intermediate (X) can beprepared using an intermediate 10

which may be formed by protecting the amino nitrogen of theaminoazobicyclooctane 5 with amino-protecting group P² and then removingP¹ from the nitrogen of the azabicyclooctane group. Protecting groups P¹and P² are chosen such that they are removed under different conditions.For example when P¹ is chosen as Boc, then Cbz can be used as P².Substituting the protected aminotropane 10 for intermediate (IV) in thereactions described above for the preparation of intermediate (III)provides intermediates of formula (X).

In yet another method of synthesis, compounds of formula (I) in which R³is hydrogen, represented below as formula (I′), can be prepared asillustrated in Scheme G.

Intermediate (XI) may contain a chiral center, as shown explicitly forthe protected oxirane intermediate (VII).

Typically, intermediate (IV) is contacted with between about 1 and about2 equivalents of the oxirane intermediate (XI) in a polar diluent, suchas ethanol to form the product (I′). Intermediate (IV) can be suppliedin salt form in which case a slight molar excess of alkaline base isincluded in the reaction mixture prior to the addition of the oxirane.The reaction is typically conducted at a temperature of about 60° C. toabout 100° C. for between about 1 and about 3 hours, or until thereaction is substantially complete. The product can be isolated bycrystallization from an inert diluent as the free base or as an acidsalt.

Intermediates of formula (XI) can be prepared by reaction of the oxiraneintermediate 1, illustrated in Scheme C, with the secondary amineHNR⁴R⁵. Typically, an aqueous solution of the amine HNR⁴R⁵ containingabout 1 equivalent of a base, such as sodium hydroxide, lithiumhydroxide, cesium hydroxide, or potassium hydroxide, is contacted withbetween about 1.5 and about 2.5 equivalents of the oxiraneintermediate 1. The reaction is typically conducted at a temperature ofbetween about 0° C. and about 10° C. for between about 12 and about 30hours, or until the reaction is substantially complete.

The quinolinone carboxylic acid (VIII) is readily prepared by proceduressimilar to those reported in the literature in Suzuki et al,Heterocycles, 2000, 53, 2471-2485 and described in the examples below.

The reagents L′-R², L′-R³, L-R⁴, H₂NR⁵, and HNR⁴R⁵ are availablecommercially or are readily prepared by standard procedures from commonstarting materials.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of the invention orintermediates thereto are described in the examples below.

Accordingly, in a method aspect, the invention provides a process forpreparing a compound of formula (I), or a salt or stereoisomer thereof,the process comprising:

(a) reacting a compound of formula (III):

with compound of the formula L-R⁴ wherein L is a leaving group, or L-R⁴represents HO—C(O)R⁷; or

(b) reacting a compound of formula (VIII):

with a compound of formula (IX):

to provide a compound of formula (I), or a salt or stereoisomer thereof.

The invention further provides a compound of formula (III), or a salt orstereoisomer or protected derivative thereof, wherein R¹, R², R³, and R⁵are defined as in formula (I).

In an additional method aspect, the invention provides a process forpreparing a compound of formula (I′) wherein R¹, R², R⁴, and R⁵ aredefined as in formula (I), or a salt or stereoisomer thereof, theprocess comprising reacting a compound of formula (IV):

or a salt thereof with a compound of formula (XI):

to provide a compound of formula (I′) or a salt or stereoisomer thereof.

Pharmaceutical Compositions

The quinolinone-carboxamide compounds of the invention are typicallyadministered to a patient in the form of a pharmaceutical composition.Such pharmaceutical compositions may be administered to the patient byany acceptable route of administration including, but not limited to,oral, rectal, vaginal, nasal, inhaled, topical (including transdermal)and parenteral modes of administration.

Accordingly, in one of its compositions aspects, the invention isdirected to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and a therapeuticallyeffective amount of a compound of formula (I) or a pharmaceuticallyacceptable salt thereof. Optionally, such pharmaceutical compositionsmay contain other therapeutic and/or formulating agents if desired.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the present inventionor a pharmaceutically-acceptable salt thereof. Typically, suchpharmaceutical compositions will contain from about 0.1 to about 95% byweight of the active agent; preferably, from about 5 to about 70% byweight; and more preferably from about 10 to about 60% by weight of theactive agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the ingredients for such compositionsare commercially-available from, for example, Sigma, P.O. Box 14508, St.Louis, Mo. 63178. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, such as microcrystallinecellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical compositions.

The pharmaceutical compositions of the invention are typically preparedby thoroughly and intimately mixing or blending a compound of theinvention with a pharmaceutically-acceptable carrier and one or moreoptional ingredients. If necessary or desired, the resulting uniformlyblended mixture can then be shaped or loaded into tablets, capsules,pills and the like using conventional procedures and equipment.

The pharmaceutical compositions of the invention are preferably packagedin a unit dosage form. The term “unit dosage form” refers to aphysically discrete unit suitable for dosing a patient, i.e., each unitcontaining a predetermined quantity of active agent calculated toproduce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such unit dosage formsmay be capsules, tablets, pills, and the like.

In a preferred embodiment, the pharmaceutical compositions of theinvention are suitable for oral administration. Suitable pharmaceuticalcompositions for oral administration may be in the form of capsules,tablets, pills, lozenges, cachets, dragees, powders, granules; or as asolution or a suspension in an aqueous or non-aqueous liquid; or as anoil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup;and the like; each containing a predetermined amount of a compound ofthe present invention as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof the invention will typically comprise a compound of the presentinvention as the active ingredient and one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate. Optionally or alternatively, such solid dosageforms may also comprise: (1) fillers or extenders, such as starches,microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants,such as glycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and/or sodium carbonate; (5) solution retarding agents, such asparaffin; (6) absorption accelerators, such as quaternary ammoniumcompounds; (7) wetting agents, such as cetyl alcohol and/or glycerolmonostearate; (8) absorbents, such as kaolin and/or bentonite clay; (9)lubricants, such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and/or mixtures thereof;(10) coloring agents; and (11) buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of the invention. Examples ofpharmaceutically-acceptable antioxidants include: (1) water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal-chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like. Coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate (CAP), polyvinyl acetatephthalate (PVAP), hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate(CAT), carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), and the like.

If desired, the pharmaceutical compositions of the present invention mayalso be formulated to provide slow or controlled release of the activeingredient using, by way of example, hydroxypropyl methyl cellulose invarying proportions; or other polymer matrices, liposomes and/ormicrospheres.

In addition, the pharmaceutical compositions of the present inventionmay optionally contain opacifying agents and may be formulated so thatthey release the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed maimer.Examples of embedding compositions which can be used include polymericsubstances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Such liquid dosage formstypically comprise the active ingredient and an inert diluent, such as,for example, water or other solvents, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (esp., cottonseed, groundnut, corn, germ,olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Alternatively, the pharmaceutical compositions of the invention areformulated for administration by inhalation. Suitable pharmaceuticalcompositions for administration by inhalation will typically be in theform of an aerosol or a powder. Such compositions are generallyadministered using well-known delivery devices, such as a metered-doseinhaler, a dry powder inhaler, a nebulizer or a similar delivery device.

When administered by inhalation using a pressurized container, thepharmaceutical compositions of the invention will typically comprise theactive ingredient and a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas.

Additionally, the pharmaceutical composition may be in the form of acapsule or cartridge (made, for example, from gelatin) comprising acompound of the invention and a powder suitable for use in a powderinhaler. Suitable powder bases include, by way of example, lactose orstarch.

The compounds of the invention can also be administered transdermallyusing known transdermal delivery systems and excipients. For example, acompound of the invention can be admixed with permeation enhancers, suchas propylene glycol, polyethylene glycol monolaurate,azacycloalkan-2-ones and the like, and incorporated into a patch orsimilar delivery system. Additional excipients including gelling agents,emulsifiers and buffers, may be used in such transdermal compositions ifdesired.

The following formulations illustrate representative pharmaceuticalcompositions of the present invention:

FORMULATION EXAMPLE A

Hard gelatin capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 50 mg Lactose (spray-dried)200 mg  Magnesium stearate 10 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then loaded into a hard gelatin capsule (260 mg of        composition per capsule).

FORMULATION EXAMPLE B

Hard gelatin capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 20 mg Starch 89 mgMicrocrystalline cellulose 89 mg Magnesium stearate  2 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then passed through a No. 45 mesh U.S. sieve and loaded into        a hard gelatin capsule (200 mg of composition per capsule).

FORMULATION EXAMPLE C

Capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 10 mg Polyoxyethylenesorbitan monooleate 50 mg Starch powder 250 mg 

-   -   Representative Procedure: The ingredients are thoroughly blended        and then loaded into a gelatin capsule (310 mg of composition        per capsule).

FORMULATION EXAMPLE D

Tablets for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 5 mg Starch 50 mg Microcrystalline cellulose 35 mg  Polyvinylpyrrolidone (10 wt. % inwater) 4 mg Sodium carboxymethyl starch 4.5 mg   Magnesium stearate 0.5mg   Talc 1 mg

-   -   Representative Procedure: The active ingredient, starch and        cellulose are passed through a No. 45 mesh U.S. sieve and mixed        thoroughly. The solution of polyvinylpyrrolidone is mixed with        the resulting powders, and this mixture is then passed through a        No. 14 mesh U.S. sieve. The granules so produced are dried at        50-60° C. and passed through a No. 18 mesh U.S. sieve. The        sodium carboxymethyl starch, magnesium stearate and talc        (previously passed through a No. 60 mesh U.S. sieve) are then        added to the granules. After mixing, the mixture is compressed        on a tablet machine to afford a tablet weighing 100 mg.

FORMULATION EXAMPLE E

Tablets for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 25 mg Microcrystallinecellulose 400 mg  Silicon dioxide fumed 10 mg Stearic acid  5 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then compressed to form tablets (440 mg of composition per        tablet).

FORMULATION EXAMPLE F

Single-scored tablets for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 15 mg Cornstarch 50 mgCroscarmellose sodium 25 mg Lactose 120 mg  Magnesium stearate  5 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and compressed to form a single-scored tablet (215 mg of        compositions per tablet).

FORMULATION EXAMPLE G

A suspension for oral administration is prepared as follows:

Ingredients Amount Compound of the invention 0.1 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum k(Vanderbilt Co.) 1.0 g Flavoring 0.035 mL Colorings 0.5 mg Distilledwater q.s. to 100 mL

Representative Procedure: The ingredients are mixed to form a suspensioncontaining 10 mg of active ingredient per 10 mL of suspension.

FORMULATION EXAMPLE H

A dry powder for administration by inhalation is prepared as follows:

Ingredients Amount Compound of the invention 1.0 mg Lactose  25 mg

-   -   Representative Procedure: The active ingredient is micronized        and then blended with lactose. This blended mixture is then        loaded into a gelatin inhalation cartridge. The contents of the        cartridge are administered using a powder inhaler.

FORMULATION EXAMPLE I

A dry powder for administration by inhalation in a metered dose inhaleris prepared as follows:

-   -   Representative Procedure: A suspension containing 5 wt. % of a        compound of the invention and 0.1 wt. % lecithin is prepared by        dispersing 10 g of active compound as micronized particles with        mean size less than 10 μm in a solution formed from 0.2 g of        lecithin dissolved in 200 mL of demineralized water. The        suspension is spray dried and the resulting material is        micronized to particles having a mean diameter less than 1.5 μm.        The particles are loaded into cartridges with pressurized        1,1,1,2-tetrafluoroethane.

FORMULATION EXAMPLE J

An injectable formulation is prepared as follows:

Ingredients Amount Compound of the invention 0.2 g Sodium acetate buffersolution (0.4 M) 40 mL HCl (0.5 N) or NaOH (0.5 N) q.s. to pH 4 Water(distilled, sterile) q.s. to 20 mL

-   -   Representative Procedure: The above ingredients are blended and        the pH is adjusted to 4±0.5 using 0.5 N HCl or 0.5 N NaOH.

FORMULATION EXAMPLE K

Capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the Invention 4.05 mg Microcrystallinecellulose (Avicel PH 103) 259.2 mg  Magnesium stearate 0.75 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then loaded into a gelatin capsule (Size #1, White, Opaque)        (264 mg of composition per capsule).

FORMULATION EXAMPLE L

Capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the Invention  8.2 mg Microcrystallinecellulose (Avicel PH 103) 139.05 mg  Magnesium stearate 0.75 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then loaded into a gelatin capsule (Size #1, White, Opaque)        (148 mg of composition per capsule).

It will be understood that any form of the compounds of the invention,(i.e. free base, pharmaceutical salt, or solvate) that is suitable forthe particular mode of administration, can be used in the pharmaceuticalcompositions discussed above.

Utility

The quinolinone-carboxamide compounds of the invention are 5-HT₄receptor agonists and therefore are expected to be useful for treatingmedical conditions mediated by 5-HT₄ receptors or associated with 5-HT₄receptor activity, i.e. medical conditions which are ameliorated bytreatment with a 5-HT₄ receptor agonist. Such medical conditionsinclude, but are not limited to, irritable bowel syndrome (IBS), chronicconstipation, functional dyspepsia, delayed gastric emptying,gastroesophageal reflux disease (GERD), gastroparesis, post-operativeileus, intestinal pseudo-obstruction, and drug-induced delayed transit.In addition, it has been suggested that some 5-HT₄ receptor agonistcompounds may be used in the treatment of central nervous systemdisorders including cognitive disorders, behavioral disorders, mooddisorders, and disorders of control of autonomic function.

In particular, the compounds of the invention increase motility of thegastrointestinal (GI) tract and thus are expected to be useful fortreating disorders of the GI tract caused by reduced motility inmammals, including humans. Such GI motility disorders include, by way ofillustration, chronic constipation, constipation-predominant irritablebowel syndrome (C-IBS), diabetic and idiopathic gastroparesis, andfunctional dyspepsia.

In one aspect, therefore, the invention provides a method of increasingmotility of the gastrointestinal tract in a mammal, the methodcomprising administering to the mammal a therapeutically effectiveamount of a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a compound of the invention.

When used to treat disorders of reduced motility of the GI tract orother conditions mediated by 5-HT₄ receptors, the compounds of theinvention will typically be administered orally in a single daily doseor in multiple doses per day, although other forms of administration maybe used. The amount of active agent administered per dose or the totalamount administered per day will typically be determined by a physician,in the light of the relevant circumstances, including the condition tobe treated, the chosen route of administration, the actual compoundadministered and its relative activity, the age, weight, and response ofthe individual patient, the severity of the patient's symptoms, and thelike.

Suitable doses for treating disorders of reduced motility of the GItract or other disorders mediated by 5-HT₄ receptors will range fromabout 0.0007 to about 20 mg/kg/day of active agent, including from about0.0007 to about 1 mg/kg/day. For an average 70 kg human, this wouldamount to from about 0.05 to about 70 mg per day of active agent.

In one aspect of the invention, the compounds of the invention are usedto treat chronic constipation. When used to treat chronic constipation,the compounds of the invention will typically be administered orally ina single daily dose or in multiple doses per day. Preferably, the dosefor treating chronic constipation will range from about 0.05 to about 70mg per day.

In another aspect of the invention, the compounds of the invention areused to treat irritable bowel syndrome. When used to treatconstipation-predominant irritable bowel syndrome, the compounds of theinvention will typically be administered orally in a single daily doseor in multiple doses per day. Preferably, the dose for treatingconstipation-predominant irritable bowel syndrome will range from about0.05 to about 70 mg per day.

In another aspect of the invention, the compounds of the invention areused to treat diabetic gastroparesis. When used to treat diabeticgastroparesis, the compounds of the invention will typically beadministered orally in a single daily dose or in multiple doses per day.Preferably, the dose for treating diabetic gastroparesis will range fromabout 0.05 to about 70 mg per day.

In yet another aspect of the invention, the compounds of the inventionare used to treat functional dyspepsia. When used to treat functionaldyspepsia, the compounds of the invention will typically be administeredorally in a single daily dose or in multiple doses per day. Preferably,the dose for treating functional dyspepsia will range from about 0.05 toabout 70 mg per day.

The invention also provides a method of treating a mammal having adisease or condition associated with 5-HT₄ receptor activity, the methodcomprising administering to the mammal a therapeutically effectiveamount of a compound of the invention or of a pharmaceutical compositioncomprising a compound of the invention.

As described above, compounds of the invention are 5-HT₄ receptoragonists. The invention further provides, therefore, a method ofagonizing a 5-HT₄ receptor in a mammal, the method comprisingadministering a compound of the invention to the mammal. In addition,the compounds of the invention are also useful as research tools forinvestigating or studying biological systems or samples having 5-HT₄receptors, or for discovering new 5-HT₄ receptor agonists. Moreover,since compounds of the invention exhibit binding selectivity for 5-HT₄receptors as compared with binding to receptors of other 5-HT subtypes,particularly 5-HT₃ receptors, such compounds are particularly useful forstudying the effects of selective agonism of 5-HT₄ receptors in abiological system or sample. Any suitable biological system or samplehaving 5-HT₄ receptors may be employed in such studies which may beconducted either in vitro or in vivo. Representative biological systemsor samples suitable for such studies include, but are not limited to,cells, cellular extracts, plasma membranes, tissue samples, mammals(such as mice, rats, guinea pigs, rabbits, dogs, pigs, etc.) and thelike.

In this aspect of the invention, a biological system or samplecomprising a 5-HT₄ receptor is contacted with a 5-HT₄ receptor-agonizingamount of a compound of the invention. The effects of agonizing the5-HT₄ receptor are then determined using conventional procedures andequipment, such as radioligand binding assays and functional assays.Such functional assays include ligand-mediated changes in intracellularcyclic adenosine monophosphate (cAMP), ligand-mediated changes inactivity of the enzyme adenylyl cyclase (which synthesizes cAMP),ligand-mediated changes in incorporation of analogs of guanosinetriphosphate (GTP), such as [³⁵S]GTPγS (guanosine5′-O-(γ-thio)triphosphate) or GTP-Eu, into isolated membranes viareceptor catalyzed exchange of GTP analogs for GDP analogs,ligand-mediated changes in free intracellular calcium ions (measured,for example, with a fluorescence-linked imaging plate reader or FLIPR®from Molecular Devices, Inc.), and measurement of mitogen activatedprotein kinase (MAPK) activation. A compound of the invention mayagonize or increase the activation of 5-HT₄ receptors in any of thefunctional assays listed above, or assays of a similar nature. A 5-HT₄receptor-agonizing amount of a compound of the invention will typicallyrange from about 1 nanomolar to about 500 nanomolar.

Additionally, the compounds of the invention can be used as researchtools for discovering new 5-HT₄ receptor agonists. In this embodiment,5-HT₄ receptor binding or functional data for a test compound or a groupof test compounds is compared to the 5-HT₄ receptor binding orfunctional data for a compound of the invention to identify testcompounds that have superior binding or functional activity, if any.This aspect of the invention includes, as separate embodiments, both thegeneration of comparison data (using the appropriate assays) and theanalysis of the test data to identify test compounds of interest.

Among other properties, compounds of the invention have been found to bepotent agonists of the 5-HT₄ receptor and to exhibit substantialselectivity for the 5-HT₄ receptor subtype over the 5-HT₃ receptorsubtype in radioligand binding assays. Further, compounds of theinvention have demonstrated superior pharmacokinetic properties in a ratmodel. Compounds of the invention are thus expected to be highlybioavailable upon oral administration. In addition, these compounds havebeen shown not to exhibit an unacceptable level of inhibition of thepotassium ion current in an in vitro voltage-clamp model using isolatedwhole cells expressing the hERG cardiac potassium channel. Thevoltage-clamp assay is an accepted pre-clinical method of assessing thepotential for pharmaceutical agents to change the pattern of cardiacrepolarization, specifically to cause, so-called QT prolongation, whichhas been associated with cardiac arrhythmia. (Cavero et al., Opinion onPharmacotherapy, 2000, 1, 947-73, Fermini et al., Nature Reviews DrugDiscovery, 2003, 2, 439-447) Accordingly, pharmaceutical compositionscomprising compounds of the invention are expected to have an acceptablecardiac profile.

There properties, as well as the utility of the compounds of theinvention, can be demonstrated using various in vitro and in vivo assayswell-known to those skilled in the art. Representative assays aredescribed in further detail in the following examples.

EXAMPLES

The following synthetic and biological examples are offered toillustrate the invention, and are not to be construed in any way aslimiting the scope of the invention. In the examples below, thefollowing abbreviations have the following meanings unless otherwiseindicated. Abbreviations not defined below have their generally acceptedmeanings.

Boc = tert-butoxycarbonyl (Boc)₂O = di-tert-butyl dicarbonate DCM =dichloromethane DMF = N,N-dimethylformamide DMSO = dimethyl sulfoxideEtOAc = ethyl acetate mCPBA = m-chlorobenzoic acid MeCN = acetonitrileMTBE = tert-butyl methyl ether PyBop =benzotriazol-1-yloxytripyrrolidino- phosphonium hexafluorophosphateR_(f) = retention factor RT = room temperature TFA = trifluoroaceticacid THF = tetrahydrofuran

Reagents (including secondary amines) and solvents were purchased fromcommercial suppliers (Aldrich, Fluka, Sigma, etc.), and used withoutfurther purification. Reactions were run under nitrogen atmosphere,unless noted otherwise. Progress of reaction mixtures was monitored bythin layer chromatography (TLC), analytical high performance liquidchromatography (anal. HPLC), and mass spectrometry, the details of whichare given below and separately in specific examples of reactions.Reaction mixtures were worked up as described specifically in eachreaction; commonly they were purified by extraction and otherpurification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by preparative HPLC: a general protocol is describedbelow. Characterization of reaction products was routinely carried outby mass and ¹H—NMR spectrometry. For NMR measurement, samples weredissolved in deuterated solvent (CD₃OD, CDCl₃, or DMSO-d₆), and 1H—NMRspectra were acquired with a Varian Gemini 2000 instrument (300 MHz)under standard observation conditions. Mass spectrometric identificationof compounds was performed by an electrospray ionization method (ESMS)with an Applied Biosystems (Foster City, Calif.) model API 150 EXinstrument or an Agilent (Palo Alto, Calif.) model 1100 LC/MSDinstrument. Water content is determined by Karl Fischer titration usinga Brinkmann (Westbury, N.Y.) Metrohm Karl Fischer Model 813 coulometer.

Example 1 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amidea. Preparation of 8-benzyl-8-azabicyclo[3.2.1]octan-3-one

Concentrated hydrochloric acid (30 mL) was added to a heterogeneoussolution of 2,5-dimethoxy tetrahydrofuran (82.2 g, 0.622 mol) in water(170 mL) while stirring. In a separate flask cooled to 0° C. (ice bath),concentrated hydrochloric acid (92 mL) was added slowly to a solution ofbenzyl amine (100 g, 0.933 mol) in water (350 mL). The2,5-dimethoxytetrahydrofuran solution was stirred for approximately 20min, diluted with water (250 mL), and then the benzyl amine solution wasadded, followed by the addition of a solution of 1,3-acetonedicarboxylicacid (100 g, 0.684 mol) in water (400 mL) and then the addition ofsodium hydrogen phosphate (44 g, 0.31 mol) in water (200 mL). The pH wasadjusted from pH 1 to pH˜4.5 using 40% NaOH. The resulting cloudy andpale yellow solution was stirred overnight. The solution was thenacidified to pH 3 from pH 7.5 using 50% hydrochloric acid, heated to 85°C. and stirred for 2 hours. The solution was cooled to room temperature,basified to pH 12 using 40% NaOH, and extracted with DCM (3×500 mL). Thecombined organic layers were washed with brine, dried (MgSO₄), filteredand concentrated under reduced pressure to produce the crude titleintermediate as a viscous brown oil (52 g).

To a solution of the crude intermediate in methanol (1000 mL) was addeddi-tert-butyl dicarbonate (74.6 g, 0.342 mol) at 0° C. The solution wasallowed to warm to room temperature and stirred overnight. The methanolwas removed under reduced pressure and the resulting oil was dissolvedin dichloromethane (1000 mL). The intermediate was extracted into 1 MH₃PO₄ (1000 mL) and washed with dichloromethane (3×250 mL) The aqueouslayer was basified to pH 12 using aqueous NaOH, and extracted withdichloromethane (3×500 mL). The combined organic layers were dried(MgSO₄), filtered and concentrated under reduced pressure to produce thetitle intermediate as a viscous, light brown oil. ¹H—NMR (CDCl₃) δ (ppm)7.5-7.2 (m, 5H, C₆H₅), 3.7 (s, 2H, CH₂Ph), 3.45 (broad s, 2H, CH—NBn),2.7-2.6 (dd, 2H, CH₂CO), 2.2-2.1 (dd, 2H, CH₂CO), 2.1-2.0 (m, 2H,CH₂CH₂), 1.6 (m, 2H, CH₂CH₂). (m/z): [M+H]⁺ calcd for C₁₄H₁₇NO 216.14;found, 216.0.

b. Preparation of 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylic acidtert-butyl ester

To a solution of 8-benzyl-8-azabicyclo[3.2.1]octan-3-one (75 g, 0.348mol) in EtOAc (300 mL) was added a solution of di-tert-butyl dicarbonate(83.6 g, 0.383 mol, 1.1 eq) in EtOAc (300 mL). The resulting solutionand rinse (100 mL EtOAc) was added to a 1 L Parr hydrogenation vesselcontaining 23 g of palladium hydroxide (20 wt. % Pd, dry basis, oncarbon, ˜50% wet with water; e.g. Pearlman's catalyst) under a stream ofnitrogen. The reaction vessel was degassed (alternating vacuum and N₂five times) and pressurized to 60 psi of H₂ gas. The reaction solutionwas agitated for two days and recharged with H₂ as needed to keep the H₂pressure at 60 psi until the reaction was complete as monitored bysilica thin layer chromatography. The black solution was then filteredthrough a pad of Celite® and concentrated under reduced pressure toyield the title intermediate quantitatively as a viscous, yellow toorange oil. It was used in the next step without further treatment. ¹HNMR (CDCl₃) δ(ppm) 4.5 (broad, 2H, CH—NBoc), 2.7 (broad, 2H, CH₂CO),2.4-2.3 (dd, 2H, CH₂CH₂), 2.1 (broad m, 2H, CH₂CO), 1.7-1.6 (dd, 2H,CH₂CH₂), 1.5 (s, 9H, (CH₃)₃COCON)).

c. Preparation of(1S,3R,SR)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylic acidtert-butyl ester

To a solution of the product of the previous step (75.4 g, 0.335 mol) inmethanol (1 L) was added ammonium formate (422.5 g, 6.7 mol), water (115mL) and 65 g of palladium on activated carbon (10% on dry basis, ˜50%wet with water; Degussa type E101NE/W) under a stream of N₂ whilestirring via mechanical stirrer. After 24 and 48 hours, additionalportions of ammonium formate (132 g, 2.1 mol) were added each time. Oncereaction progression ceased, as monitored by anal. HPLC, Celite® (>500g) was added and the resulting thick suspension was filtered and thenthe collected solid was rinsed with methanol (˜500 mL). The filtrateswere combined and concentrated under reduced pressure until all methanolhad been removed. The resulting cloudy, biphasic solution was thendiluted with 1M phosphoric acid to a final volume of ˜1.5 to 2.0 L at pH2 and washed with dichloromethane (3×700 mL). The aqueous layer wasbasified to pH 12 using 40% aq. NaOH, and extracted with dichloromethane(3×700 mL). The combined organic layers were dried over MgSO₄, filtered,and concentrated by rotary evaporation, then high-vacuum leaving 52 g(70%) of the title intermediate, commonly N-Boc-endo-3-aminotropane, asa white to pale yellow solid. The isomer ratio of endo to exo amine ofthe product was >99:1 based on ¹H—NMR analysis (>96% purity byanalytical HPLC). ¹H NMR (CDCl₃) δ (ppm) 4.2-4.0 (broad d, 2H, CHNBoc),3.25 (t, 1H, CHNH₂), 2.1-2.05 (m, 4H), 1.9 (m, 2H), 1.4 (s, 9H,(CH₃)₃OCON), 1.2-1.1 (broad, 2H). (m/z): [M+H]⁺ calcd for C₁₂H₂₂N₂O₂)227.18; found, 227.2. Analytical HPLC (isocratic method; 2:98 (A:B) to90:10 (A:B) over 5 min): retention time=3.68 min.

d. Preparation of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylicacid

First, acetone (228.2 mL, 3.11 mol) was added to a stirred suspension of2-aminophenylmethanol (255.2 g, 2.07 mol) and acetic acid (3.56 mL, 62mmol) in water (2 L) at room temperature. After 4 h, the suspension wascooled to 0° C. and stirred for an additional 2.5 h and then filtered.The solid was collected and washed with water and the wet solid cooledand dried by lyophilisation to yield2,2,-dimethyl-1,4-dihydro-2H-benzo[1,3]oxazine (332.2 g, 98%) as anoff-white solid. 1H NMR (CDCl₃; 300 MHz): 1.48 (s, 6H, C(CH ₃)₂), 4.00(bs, 1H, NH), 4.86 (s, 2H, CH ₂), 6.66 (d, 1H, ArH), 6.81 (t, 1H, ArH),6.96 (d, 1H, ArH), 7.10 (t, 1H, ArH).

A solution of 2,2,-dimethyl-1,4-dihydro-2H-benzo[1,3]oxazine (125 g,0.77 mol) in THF (1 L) was filtered through a scintillation funnel andthen added dropwise via an addition funnel, over a period of 2.5 h, to astirred solution of 1.0 M LiAlH₄ in THF (800 mL) at 0° C. The reactionwas quenched by slow portionwise addition of Na₂SO₄.10H₂O (110 g), overa period of 1.5 h, at 0° C. The reaction mixture was stirred overnight,filtered and the solid salts were washed thoroughly with THF. Thefiltrate was concentrated under reduced pressure to yield2-isopropylaminophenylmethanol (120 g, 95%) as a yellow oil. 1H NMR(CDCl₃; 300 MHz): 1.24 (d, 6H, CH(CH ₃)₂), 3.15 (bs, 1H, OH), 3.61(sept, 1H, CH(CH₃)₂), 4.57 (s, 2H, CH ₂), 6.59 (t, 1H, ArH), 6.65 (d,1H, ArH), 6.99 (d, 1H, ArH), 7.15 (t, 1H, ArH).

Manganese dioxide (85% 182.6 g, 1.79 mol) was added to a stirredsolution of 2-isopropylaminophenylmethanol (118 g, 0.71 mol) in toluene(800 mL) and the reaction mixture was heated to 117° C. for 4 h. Thereaction mixture was allowed to cool to room temperature overnight andthen filtered through a pad of Celite which was eluted with toluene. Thefiltrate was concentrated under reduced pressure to yield2-isopropylaminobenzaldehyde (105 g, 90%) as an orange oil. 1H NMR(CDCl₃; 300 MHz): 1.28 (d, 6H, CH(CH ₃)₂), 3.76 (sept, 1H, CH(CH₃)₂),6.65 (t, 1H, ArH), 6.69 (d, 1H, ArH), 7.37 (d, 1H, ArH), 7.44 (t, 1H,ArH), 9.79 (s, 1H, CHO).

2,2-Dimethyl-[1,3]dioxane-4,6-dione, commonly Meldrum's acid, (166.9 g,1.16 mol) was added to a stirred solution of2-isopropylaminobenzaldehyde (105 g, 0.64 mol), acetic acid (73.6 mL,1.29 mol) and ethylenediamine (43.0 mL, 0.64 mol) in methanol (1 L) at0° C. The reaction mixture was stirred for 1 h at 0° C. and then at roomtemperature overnight. The resulting suspension was filtered and thesolid washed with methanol and collected to yield the titleintermediate, 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid(146 g, 98%) as an off-white solid. 1H NMR (CDCl₃; 300 MHz): 1.72 (d,6H, CH(CH ₃)₂), 5.50 (bs, 1H, CH(CH₃)₂), 7.44 (t, 1H, ArH), 7.75-7.77(m, 2H, ArH), 7.82 (d, 1H, ArH), 8.89 (s, 1H, CH).

e. Preparation of(1S,3R,5R)-3-[1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-8-azabicyclo[3.2.1]octane-8-carboxylicacid tert-butyl ester

Thionyl chloride (36.6 mL, 0.52 mol) was added to a stirred suspensionof 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid (80 g, 0.35mol) in toluene (600 mL) at 85° C. and the reaction mixture then heatedto 95° C. for 2 h. The reaction mixture was cooled to room temperatureand then added over 25 min to a vigorously stirred biphasic solution of(1S,3R,5R)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylic acidtert-butyl ester (78.2 g, 0.35 mol) and sodium hydroxide (69.2 g, 1.73mol) in toluene/water (1:1) (1 L) at ° C. After 1 h, the layers wereallowed to separate and the organic phase concentrated under reducedpressure. The aqueous phase was washed with EtOAc (1 L) and then (500mL) and the combined organic extracts used to dissolve the concentratedorganic residue. This solution was washed with 1M H₃PO₄ (500 mL), sat.aq. NaHCO₃ (500 mL) and brine (500 mL), dried over MgSO₄, filtered andconcentrated under reduced pressure to yield the title intermediate(127.9 g, approx. 84%) as a yellow solid. ¹H NMR (CDCl₃): 1.47 (s, 9H),1.67 (d, 6H), 1.78-1.84 (m, 2H), 2.04-2.18 (m, 6H), 4.20-4.39 (m, 3H),5.65 (bs, 1H), 7.26 (dd. 1H), 7.63 (m, 2H), 7.75 (dd, 1H), 8.83 (s, 1H),10.63 (d, 1H).

f. Preparation of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylicacid {(1S,3R,5R)-8-azabicyclo[3.2.1]oct-3-yl}amide

TFA (300 mL) was added to a stirred solution of the product of theprevious step (127.9 g) in CH₂Cl₂ (600 mL) at 0° C. The reaction mixturewas warmed to room temperature and stirred for 1 h and then concentratedunder reduced pressure. The oily brown residue was then poured into avigorously stirred solution of ether (3 L) and a solid precipitateformed immediately. The suspension was stirred overnight and then thesolid collected by filtration and washed with ether to yield the titleintermediate as its trifluoroacetic acid salt (131.7 g; 86% over twosteps) as a light yellow solid. ¹H NMR (CDCl₃): 1.68 (d, 6H), 2.10 (d,2H), 2.33-2.39 (m, 4H), 2.44-2.61 (m, 2H), 4.08 (bs, 2H), 4.41 (m, 1H),5.57 (bs, 1H), 7.31 (m. 1H), 7.66 (m, 2H), 7.77 (d, 1H), 8.83 (s, 1H),9.38 (bd, 2H), 10.78 (d, 1H).

g. Preparation of3-hydroxy-3′-{[1-isopropyl-2-oxo-1,2-dihydroquinolin-3-yl)carbonyl]amino}spiro[azetidine-1,8′-(1S,3R,5R)-8-azabicyclo[3.2.1]octane(Intermediate (V) with R¹═H. R²=isopropyl)

2-Bromomethyloxirane (10.72 mL, 129.5 mmol) was added to a stirredsolution of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-aza-bicyclo[3.2.1]oct-3-yl}amide trifluoroacetic acid salt(14.65 g, 43.2 mmol) in ethanol (150 mL) at room temperature. Thereaction mixture was stirred for 36 h, at which time a solid precipitateformed. The solid was collected by filtration and washed with ethanol(70 mL) to yield the title intermediate as the bromide salt (8.4 g).(m/z): [M]⁺ calcd for C₂₃H₃₀N₃O₃ 396.23; found, 396.5. Retention time(anal. HPLC: 2-50% MeCN/H₂O over 5 min)=4.13 min.

h. Preparation of 1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylicacid{(1S,3R,5R)-8-[2-hydroxy-3-methylaminopropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

3-Hydroxy-3′-{[1-isopropyl-2-oxo-1,2-dihydroquinolin-3-yl)carbonyl]amino}spiro[azetidine-1,8′-(1S,3R,5R)-8-aza-bicyclo[3.2.1]octanebromide (678 mg, 1.4 mmol) was dissolved in ethanol (10 mL), and thenmethylamine (41% solution in water) (510 μL, 8.0 mmol) was added. Themixture was heated at 80° C. for 16 h, and then concentrated underreduced pressure to give the title intermediate as a crude oil, whichwas used directly in the following step.

i. Synthesis of 1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylicacid{(1S,3R,5R)-8-[2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

The product of the previous step was dissolved in dichloromethane (10mL), and then 1,8-diazabicyclo[5.4.0]undec-7-ene (763 μL, 5.1 mmol) wasadded, and the mixture was stirred under nitrogen and cooled to 0° C.Methanesulfonylchloride (132 μL, 1.7 mmol) was added and the mixture wasstirred at 0° C. for 30 min. The reaction was quenched by the additionof water, and concentrated to dryness under reduced pressure. Theproduct was taken up in acetic acid/water (1:1) (10 mL) and purified byHPLC chromatography. The purified fractions were lyophilized yieldingthe title compound as the trifluoroacetic acid salt (340 mg). (m/z):[M+H]⁺ calcd for C₂₅H₃₆N₄O₅S, 505.25; found, 505.4. Retention time(anal. HPLC: 2-50% MeCN/H₂O over 5 min)=4.17 min.

Example 2 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[2-methoxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amidea. Preparation of3-methoxy-3′-{[1-isopropyl-2-oxo-1,2-dihydroquinolin-3-yl)carbonyl]amino}spiro[azetidine-1,8′-(1S,3R,5R)-8-aza-bicyclo[3.2.1]octane(Intermediate (V′!) with R¹═H, R²=isopropyl, R³=methyl)

Potassium-tert-butoxide (1.63 g, 14.5 mmol) was added to a stirredsuspension of3-hydroxy-3′-{[1-isopropyl-2-oxo-1,2-dihydroquinolin-3-yl)carbonyl]amino}spiro[azetidine-1,8′-(1S,3R,5R)-8-azabicyclo[3.2.1]octanebromide (3.45 g, 7.25 mmol) in dichloromethane (100 mL) at roomtemperature. After 2 min, methyl iodide (0.477 mL, 7.61 mmol) was addedto the reaction mixture. After 30 min, water (2 mL) was added to quenchthe reaction and the reaction mixture concentrated under reducedpressure. The residue was dissolved in a minimal volume of aceticacid/water (1:1) and purified by preparative HPLC to yield the titleintermediate as a trifluoroacetic acid salt (2.1 g). (m/z): [M]⁺ calcdfor C₂₄H₃₂N₃O₃, 410.24; found 410.5. Retention time (anal. HPLC: 2-50%MeCN/H₂O over 5 min)=4.36min.

b. Preparation of 1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylicacid{(1S,3R,5R)-8-[2-methoxy-3-methylaminopropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

3-Methoxy-3′-{[1-isopropyl-2-oxo-1,2-dihydroquinolin-3-yl)carbonyl]amino}spiro[azetidine-1,8′-(1S,3R,5R)-8-aza-bicyclo[3.2.1]octanetrifluoroacetic acid salt (410 mg, 0.84 mmol) was dissolved in ethanol(10 mL), and then methylamine (41% solution in water, 320 μL, 5 mmol)was added. The mixture was heated at 80° C. for 16 h, and thenconcentrated under reduced pressure to give the product as a crude oilwhich was used directly in the following step.

c. Synthesis of 1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylicacid{(1S,3R,5R)-8-[2-methoxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

The product of the previous step (53.6 mg, 0.12 mmol) was dissolved indichloromethane (1.0 mL), and then 1,8-diazabicyclo[5.4.0]undec-7-ene(89.7 μL, 0.6 mmol) was added, and the mixture was stirred undernitrogen and cooled to 0° C. Methanesulfonylchloride (18.6 μL, 0.24mmol) was added and the mixture was stirred at 0° C. for 30 min. Themixture was quenched by the addition of water, and concentrated todryness under reduced pressure. The product was taken up in aceticacid/water (1:1) (1.5 mL) and purified by HPLC chromatography. Thepurified fractions were lyophilized to give the title compound as thetrifluoroacetic acid salt (45.8 mg). (m/z): [M+H]⁺ calcd forC₂₆H₃₈N₄O₅S, 519.27; found 519.2. Retention time (anal. HPLC: 5-40%MeCN/H₂O over 4 min)=2.72 min.

Example 3 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-(methanesulfonyl-pyridin-3-ylmethyl-amino)-2-methoxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amidea. Preparation of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylicacid{(1S,3R,5R)-8-{2-methoxy-3-[(pyridin-3-ylmethyl)amino]propyl}-8-azabicyclo[3.2.1]oct-3-yl}amide

3-Methoxy-3′-{[1-isopropyl-2-oxo-1,2-dihydroquinolin-3-yl)carbonyl]amino}spiro[azetidine-1,8′-(1S,3R,5R)-8-aza-bicyclo[3.2.1]octane(410 mg, 0.84 mmol) was dissolved in ethanol (10 mL), and then3-aminomethylpyridine (153 μL, 1.5 mmol) was added. The mixture washeated at 60° C. for 16 h, and then concentrated under reduced pressureto give the title intermediate as a crude oil which was used directly inthe next step.

b. Synthesis of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-(methanesulfonyl-pyridin-3-ylmethyl-amino)-2-methoxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

The product of the previous step (102.6 mg, 0.2 mmol) was dissolved indichloromethane (1.0 mL ) and then 1,8-diazabicyclo[5.4.0]undec-7-ene(119.6 μL, 0.8 mmol) was added, and the mixture was stirred undernitrogen and cooled to 0° C. Methanesulfonylchloride (30.1 μL, 0.4 mmol)was added and the mixture was stirred at 0° C. for 30 min. The reactionwas quenched by the addition of water, and the mixture was concentratedto dryness under reduced pressure. The product was taken up in aceticacid/water (1:1) (1.5 mL) and purified by HPLC chromatography. Thepurified fractions were lyophilized to give the title compound as thetrifluoroacetic acid salt (63.5 mg). (m/z): [M+H]⁺ calcd forC₃₁H₄₁N₅O₅S, 596.29; found 596.2. Retention time (anal. HPLC: 5-40%MeCN/H₂O over 4 min)=2.35 min.

Example 4 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[2-hydroxy-3-methanesulfonylamino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amidea. Preparation of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylicacid{(1S,3R,5R)-8-[3-(1,3-dioxo-1,3-dihydroisoindol-2-yl)-2-hydroxypropy]-8-azabicyclo[3.2.1]oct-3-yl}amide

1-Isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-aza-bicyclo[3.2.1]oct-3-yl}amide (3.39 g, 10 mmol) wasdissolved in ethanol (40 mL), and then2-oxiranylmethylisoindole-1,3-dione, commonly, epoxypropylphthalimide,(3.05 g, 15 mmol) was added. The mixture was heated at 80° C. for 36 h,cooled, and then concentrated under reduced pressure. The resulting oilwas flash chromatographed (SiO₂, eluting with a 9:1 solution ofdichloromethane/methanol) to give the title intermediate (4.95 g) as awhite solid which was used directly in the next step.

b. Preparation of 1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylicacid{(1S,3R,5R)-8-[2-hydroxy-3-aminopropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

The product of the previous step (4.95 g, 9.13 mmol) was dissolved inethanol (40 mL), and then hydrazine (860 μL, 27.4 mmol) was added. Themixture was refluxed for 16 h, and then cooled to room temperature. Themixture was filtered, and the filtrate concentrated to give the titleintermediate as a crude oil, which was used directly without furtherpurification.

c. Synthesis of 1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylicacid{(1S3R,5R)-8-[2-hydroxy-3-methanesulfonylamino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

The product of the previous step (82 mg, 0.2 mmol) was dissolved indichloromethane (1.0 mL), then 1,8-diazabicyclo[5.4.0]undec-7-ene (60μL, 0.4 mmol) was added, and the mixture was stirred under nitrogen andcooled to −78° C. Methanesulfonylchloride (15.5 μL, 0.2 mmol) was addedand the mixture was stirred and allowed to warm to room temperature over30 min. The reaction was quenched by the addition of water, and themixture was concentrated to dryness under reduced pressure. The productwas taken up in acetic acid/water (1:1) (1.5 mL) and purified by HPLCchromatography. The purified fractions were lyophilized to give thetitle compound as the trifluoroacetic acid salt (70.7 mg). (m/z): [M+H]⁺calcd for C₂₄H₃₄N₄O₅S, 491.23; found 491.2. Retention time (anal. HPLC:5-40% MeCN/H₂O over 4 min)=2.43 min.

Example 5 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-(methanesulfonyl-pyridin-3-ylmethyl-amino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amidea. Preparation of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylicacid{(1S3R,5R)-8-{2-hydroxy-3-[(pyridin-3-ylmethyl)amino]propyl}-8-azabicyclo[3.2.1]oct-3-yl}amide

3-Hydroxy-3′-{[1-isopropyl-2-oxo-1,2-dihydroquinolin-3-yl)carbonyl]amino}spiro[azetidine-1,8′-(1S,3R,5R)-8-aza-bicyclo[3.2.1]octanebromide (505 mg, 1.27 mmol) was dissolved in ethanol (10 mL), and then3-(aminomethyl)-pyridine (193 μL, 1.9 mmol) was added. The mixture washeated at 80° C. for 16 h, and then concentrated under reduced pressureto give the title intermediate as a crude oil which was used directly inthe following step.

b. Synthesis of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-(methanesulfonyl-pyridin-3-ylmethyl-amino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

The product of the previous step (42.5 mg, 0.08 mmol) was dissolved indichloromethane (1.0 mL), 1,8-diazabicyclo[5.4.0]undec-7-ene (74.8 μL,0.5 mmol) was added, and the mixture was stirred under nitrogen andcooled to 0° C. Methanesulfonylchloride (6.1 μL, 0.08 mmol) was addedand the mixture was stirred at 0° C. for 30 min. The reaction wasquenched by the addition of water, and concentrated to dryness underreduced pressure. The product was taken up in acetic acid/water (1:1)(1.5 mL) and purified by HPLC chromatography. The purified fractionswere lyophilized to give the title compound as the trifluoroacetic acidsalt (22.8 mg). (m/z): [M+H]⁺ calcd for C₃₀H₃₉N₅O₅S, 582.28; found582.2. Retention time (anal. HPLC: 5-40% MeCN/H₂O over 4 min)=2.78 min.

Example 6 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amidea. Preparation of (S)-1-(benzyl-methyl-amino)-3-chloropropan-2-ol

N-Benzylmethylamine (13.95 mL, 108.1 mmol) and(S)-2-chloromethyloxirane, commonly (S)-epichlorohydrin (8.48 mL, 108.1mmol) were dissolved in hexane (40 mL), and stirred for 16 h. Thesolution was then flash chromatographed (SiO₂, eluting with 10%methanol/90% dichloromethane). Fractions containing product wereconcentrated to give the title intermediate as an oil (19.7 g). ¹H—NMR(DMSO d6, 299.96 MHz): δ (ppm) 2.01 (s, 3H), 2.2-2.4 (m, 2H), 3.21-3.5(m, 3H), 3.53-3.6 (m, 1H), 3.65-3.75 (m, 1H), 4.95 (d, 1H), 7.0-7.25 (m,5H). (m/z): [M+H]⁺ calcd for C₁₁H₁₆ClNO, 214.10; found 214.1.

b. Preparation of ((LD-3-chloro-2-hydroxypropyl)methylcarbamic acidtert-butyl ester

(S)-1-(benzyl-methyl-amino)-3-chloropropan-2-ol (8.4 g, 39.3 mmol) wasdissolved in ethyl acetate (75 mL), and then di-tert-butyl dicarbonate(9.3 g, 43.23 mmol) and palladium hydroxide (2.5 g) were added. Themixture was shaken for 12 h under hydrogen (60 atm). The mixture wasfiltered through a bed of Celite®, and concentrated to dryness underreduced pressure. The resulting oil was filtered through silica, elutingwith hexane, followed by dichloromethane, followed by diethyl ether. Theether layer was concentrated to give the title intermediate as an oil(7.1 g). ¹H—NMR (DMSO d₆, 299.96 MHz): δ (ppm) 1.35-1.46 (s, 9H),2.81-2.85 (s, 3H), 2.95-3.1 (m, 1H), 3.3-3.6 (m, 3H), 3.67-3.85 (m, 1H),5.25-5.4 (m, 1H). (m/z): [M+H-Boc]⁺ calcd for C₉H₁₈ClNO₃, 123.10; found123.1.

c. Preparation of((R)-2-hydroxy-3-{3-[(1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-(1S,3R,5R)-8-azabicyclo[3.2.1]oct-8-yl}propyl)methylcarbamicacid tert-butyl ester

((S)-3-chloro-2-hydroxypropyl)methylcarbamic acid tert-butyl ester (335mg, 1.5 mmol) and 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylicacid {(1S,3R,5R)-8-aza-bicyclo[3.2.1]oct-3-yl}amide (339 mg, 1.0 mmol),were dissolved in methanol (5 mL) and then N,N-diisopropylethylamine(523 μL, 3.0 mmol) was added. The mixture was heated at 80° C. for 16h,and then concentrated under reduced pressure. The resulting oil wasflash chromatographed (SiO₂, eluting with 10% methanol/90%dichloromethane). Fractions containing product were concentrated to givethe title intermediate as a white solid (0.5 g). (m/z): [M+H]⁺ calcd forC₂₉H₄₂N₄O₅, 527.33; found 527.6. Retention time (anal. HPLC: 2-50%MeCN/H₂O over 5 min)=4.75 min.

d. Preparation of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylicacid{(1S,3R,5R)-8-[(S)-2-hydroxy-3-methylaminopropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

((R)-2-hydroxy-3-{3-[(1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-(1S,3R,5R)-8-azabicyclo[3.2.1]oct-8-yl}propyl)methylcarbamicacid tert-butyl ester (575 mg, 1.09 mmol) was dissolved indichloromethane (5 mL) and then trifluoroacetic acid (5 mL) was addedslowly. The mixture was stirred for 30 min, and then concentrated underreduced pressure. The resulting oil was triturated with diethyl ether,and then filtered. The precipitate was dried under vacuum to give thetitle intermediate as the trifluoroacetic acid salt (0.68 g). (m/z):[M+H]⁺ calcd for C₂₄H₃₄N₄O₃, 427.27; found 427.2. Retention time (anal.HPLC: 2-50% MeCN/H₂O over 5 min)=3.40 min.

e. Synthesis of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amide

1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(S)-2-hydroxy-3-methylaminopropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide(1.03 g, 1.57 mmol) was dissolved in dichloromethane (6.0 mL),1,8-diazabicyclo[5.4.0]undec-7-ene (747 μL, 5.0 mmol) was added, and themixture was stirred under nitrogen and cooled to 0° C.Methanesulfonylchloride (124.4 μL, 1.6 mmol) was added and the mixturewas stirred at 0° C. for 30 min. The reaction was quenched by theaddition of water, and the mixture was concentrated to dryness underreduced pressure. The product was taken up in acetic acid/water (1:1) (5mL) and purified by HPLC chromatography. The purified fractions werelyophilized to give the title compound as the trifluoroacetic acid salt(0.38 g).

(m/z): [M+H]⁺ calcd for C₂₅H₃₆N₄O₅S, 505.25; found 505.4. Retention time(anal. HPLC: 2-50% MeCN/H₂O over 5 min)=4.17 min. Free base: ¹H—NMR(DMSO d6, 299.96 MHz): δ (ppm) 1.40-1.68 (d, 6H), 1.81-2.02 (br s, 4H),2.02-2.18 (br m, 2H), 2.22-2.36 (d, 2H), 2.78-2.90 (2 s, 6H), 2.91-3.04(m, 1H), 3.10-3.30 (m, 4H), 3.61-3.78 (br s, 1H), 4.02-4.17 (m, 1H),4.71-4.79 (br s, 1H), 5.2-5.8 (br s, 1H), 7.3-7.4 (t, 1H), 7.67-7.78 (t,1H), 7-82-7.94 (d, 1H), 7.98-8.02 (d, 1H), 8.80 (s, 1H), 10.37-10.40 (d,NH).

Example 7 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(S)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amide

Following the procedure of Example 6, with the substitution of(R)-2-chloromethyloxirane for (S)-2-chloromethyloxirane in step a, thefollowing intermediates and the title compound were prepared.

(R)-1-(benzyl-methyl-amino)-3-chloropropan-2-ol: ¹H—NMR (DMSO d₆, 299.96MHz): δ (ppm) 2.01 (s, 3H), 2.2-2.4 (m, 2H), 3.21-3.5 (m, 3H), 3.53-3.6(m, 1H), 3.65-3.75 (m, 1H), 4.95 (d, 1H), 7.0-7.25 (m, 5H). (m/z):[M+H]⁺ calcd for C₁₁H₁₆ClNO, 214.10; found 214.1.

((R)-3-chloro-2-hydroxy-propyl)methylcarbamic acid tert-butyl ester:¹H—NMR (DMSO d₆, 299.96 MHz): δ (ppm) 1.35-1.46 (s, 9H), 2.81-2.85 (s,3H), 2.95-3.1 (m, 1H), 3.3-3.6 (m, 3H), 3.67-3.85 (m, 1H), 5.25-5.4 (m,1H). (m/z): [M+H-Boc]⁺ calcd for C₉H₁₈ClNO₃, 123.10; found 123.1.

((S)-2-hydroxy-3-{3-[(1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-(1S,3R,5R)-8-azabicyclo[3.2.1]oct-8-yl}propyl)methylcarbamicacid tert-butyl ester: (m/z): [M+H]⁺ calcd for C₂₉H₄₂N₄O₅, 527.33; found527.6. Retention time (anal. HPLC: 2-50% MeCN/H₂O over 5 min)=4.75 min.

1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-methylaminopropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide:(m/z): [M+H]⁺ calcd for C₂₄H₃₄N₄O₃, 427.27; found 427.2. Retention time(anal. HPLC: 2-50% MeCN/H₂O over 5 min)=3.40 min.

1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(S)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amide:(m/z): [M+H]⁺ calcd for C₂₅H₃₆N₄O₅S, 505.25; found 505.4. Retention time(anal. HPLC: 2-50% MeCN/H₂O over 5 min)=4.17 min.

Example 8 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[2-hydroxy-3-[methyl-(pyridine-4-carbonyl)amino]propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[2-hydroxy-3-methylaminopropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide(71 mg, 0.17 mmol) was dissolved in DMF (0.5 mL), and thenN,N-diisopropylethylamine (88.9 μL, 0.51 mmol) was added. Next, asolution of isonicotinic acid (41.8 mg, 0.34 mmol) and PyBOP (177 mg,0.34 mmol) in DMF was added. The resulting mixture was shaken at roomtemperature for 30 min, and then concentrated to dryness under reducedpressure. The product was taken up in acetic acid/water (1:1) (1.5 mL)and purified by HPLC chromatography. The purified fractions werelyophilized to give the title compound as the trifluoroacetic acid salt(30.8 mg). (m/z): [M+H]⁺ calcd for C₃₀H₃₇N₅O₄, 532.29; found 532.7.Retention time (anal. BPLC: 5-40% MeCN/H₂O over 4 min)=2.11 min.

Example 9 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[2-hydroxy-3-[(pyridine-4-carbonyl)amino]propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

1-Isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[2-hydroxy-3-aminopropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide(82.4 mg, 0.2 mmol) was dissolved in DMF (0.5 mL), and thenN,N-diisopropylethylamine (69.7 μL, 0.4 mmol) was added. Next, asolution of isonicotinic acid (49.2 mg, 0.4 mmol) and PyBOP (208.2 mg,0.4 mmol) in DMF was added. The resulting mixture was shaken at roomtemperature for 30 min, and then concentrated to dryness under reducedpressure. The product was taken up in acetic acid/water (1:1) (1.5 mL)and purified by HPLC chromatography. The purified fractions werelyophilized to give the title compound as the trifluoroacetic acid salt(82.1 mg). (m/z): [M+H]⁺ calcd for C₂₉H₃₅N₅O₄, 518.28; found 518.2.Retention time (anal. HPLC: 5-40% MeCN/H₂O over 4 min)=2.20 min.

Example 10 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-(acetyl-methyl-amino)-2-methoxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

1-Isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[2-methoxy-3-methylaminopropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide(53.6 mg, 0.12 mmol) was dissolved in DMF (1.0 mL), and thenN,N-diisopropylethylamine (104. μL, 0.6 mmol) was added. The mixture wasstirred under nitrogen and cooled to 0° C. Acetyl chloride (17.1 μL,0.24 mmol) was added and the mixture was stirred at 0° C. for 30 min,and then concentrated to dryness under reduced pressure. The product wastaken up in acetic acid/water (1:1) (1.5 mL) and purified by HPLCchromatography. The purified fractions were lyophilized to give thetitle compound as the trifluoroacetic acid salt (40.4 mg). (m/z): [M+H]⁺calcd for C₂₇H₃₈N₄O₄, 483.30; found 483.2. Retention time (anal. HPLC:5-40% MeCN/H₂O over 4 min)=2.54 min.

Example 11 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[2-methoxy-3-[methyl-(pyridine-4-carbonyl)amino]propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

1-Isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[2-methoxy-3-methylaminopropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide(53.6 mg, 0.12mmol) was dissolved in DMF (1.0 mL), and thenN,N-diisopropylethylamine (104.5 μL, 0.6 mmol) was added. The mixturewas stirred under nitrogen and cooled to 0° C. Isonicotinyl chloride(42.7 mg, 0.24 mmol) was added and the mixture was stirred at 0° C. for30 min, and then concentrated to dryness under reduced pressure. Theproduct was taken up in acetic acid/water (1:1) (1.5 mL) and purified byHPLC chromatography. The purified fractions were lyophilized to give thetitle compound as the trifluoroacetic acid salt (54.4 mg). (m/z): [M+H]⁺calcd for C₃₁H₃₉N₅O₄, 546.31; found 546.2. Retention time (anal. HPLC:5-40% MeCN/H₂O over 4 min)=2.29 min.

Example 12 Synthesis of1-isopropyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-(acetyl-pyridin-3-ylmethyl-amino)-2-methoxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

1-Isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid(8-{2-methoxy-3-[(pyridin-3-ylmethyl)amino]propyl}-8-azabicyclo[3.2.1]oct-3-yl)amide(102.6 mg, 0.2 mmol) was dissolved in DMF (1.0 mL), and thenN,N-diisopropylethylamine (139.4 μL, 0.8 mmol) was added. The mixturewas stirred under nitrogen and cooled to 0° C. Acetyl chloride (28.5 μL,0.4 mmol) was added and the mixture was stirred at 0° C. for 30 min, andthen concentrated to dryness under reduced pressure. The product wastaken up in acetic acid/water (1:1) (1.5 mL) and purified by HPLCchromatography. The purified fractions were lyophilized to give thetitle compound as the trifluoroacetic acid salt (33.2 mg). (m/z): [M+H]⁺calcd for C₃₂H₄₁N₅O₄, 560.33; found 560.4. Retention time (anal. HPLC:5-40% MeCN/H₂O over 4 min)=2.27 min.

Example 13 Synthesis of1-isopropyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-acetylamino-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

1-Isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[2-hydroxy-3-aminopropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide(82.4 mg, 0.2 mmol) was dissolved in DMF (0.5 mL), and thenN,N-diisopropylethylamine (69.7 μL, 0.4 mmol) was added. Next, asolution of acetic acid (22.7 μL, 0.4 mmol) and PyBOP (208.2 mg, 0.4mmol) in DMF was added. The resulting mixture was shaken at roomtemperature for 30 min, and then concentrated to dryness under reducedpressure. The product was taken up in acetic acid/water (1:1) (1.5 mL)and purified by HPLC chromatography. The purified fractions werelyophilized to give the title compound as the trifluoroacetic acid salt(79.2 mg). (m/z): [M+H]⁺ calcd for C₂₅H₃₄N₄O₄, 455.27; found 455.2.Retention time (anal. HPLC: 5-40% MeCN/H₂O over 4 min)=2.33 min.

Example 14 Synthesis of1-isopropyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-(acetyl-methyl-amino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

1-Isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[2-hydroxy-3-methylaminopropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide(43 mg, 0.1 mmol) was dissolved in DMF (1.0 mL), and thenN,N-diisopropylethylamine (87.1 μL, 0.5 mmol) was added. The mixture wasstirred under nitrogen and cooled to 0° C. Acetyl chloride (17.8 μL,0.25 mmol) was added and the mixture was stirred at 0° C. for 30 min.The mixture was concentrated to dryness under reduced pressure. Theproduct was taken up in acetic acid/water (1:1) (1.5 mL) and purified byHPLC chromatography. The purified fractions were lyophilized to give thetitle intermediate as the trifluoroacetic acid salt (17.1 mg). (m/z):[M+H]⁺ calcd for C₂₆H₃₆N₄O₄, 469.28; found 469.2. Retention time (anal.HPLC: 5-40% MeCN/H₂O over 4 min)=2.49 min.

Example 15 Synthesis of1-isopropyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-(formyl-methyl-amino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

1-Isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[2-hydroxy-3-methylaminopropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide(43 mg, 0.1 mmol) was dissolved in ethyl formate (1.0 mL). The mixturewas heated at 65° C. for 16 h, and then concentrated to dryness underreduced pressure. The product was taken up in acetic acid/water (1:1)(1.5 mL) and purified by HPLC chromatography. The purified fractionswere lyophilized to give the title intermediate as the trifluoroaceticacid salt (22.7 mg). (m/z): [M+H]⁺ calcd for C₂₅H₃₄N₄O₄, 455.27; found455.2. Retention time (anal. HPLC: 5-40% MeCN/H₂O over 4 min)=2.37 min.

Example 16 Synthesis of1-isopropyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-formylamino-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

1-Isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[2-hydroxy-3-aminopropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide(41.2 mg, 0.1 mmol) was dissolved in ethyl formate (1.0 mL). The mixturewas heated at 65° C. for 16 h, and then concentrated to dryness underreduced pressure. The product was taken up in acetic acid/water (1:1)(1.5 mL) and purified by HPLC chromatography. The purified fractionswere lyophilized to give the title intermediate as the trifluoroaceticacid salt (37.5 mg). (m/z): [M+H]⁺ calcd for C₂₄H₃₂N₄O₄, 441.25; found441.2. Retention time (anal. HPLC: 5-40% MeCN/H₂O over 4 min)=2.89 min.

Example 17 Synthesis of1-isopropyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-3-(acetyl-methyl-amino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

1-Isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(S)-2-hydroxy-3-methylaminopropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide(200 mg, 0.3 mmol) was dissolved in DMF (1.0 mL), and thenN,N-diisopropylethylamine (160.3 μL, 0.92 mmol) was added. Next, asolution of acetic acid (17.3 μL, 0.3 mmol) and PyBOP (159 mg, 0.3 mmol)in DMF was added. The resulting mixture was shaken at room temperaturefor 30 min, and then concentrated to dryness under reduced pressure. Theproduct was taken up in acetic acid/water (1:1) (1.5 mL) and purified byHPLC chromatography. The purified fractions were lyophilized to give thetitle intermediate as the trifluoroacetic acid salt (130 mg). (m/z):[M+H]⁺ calcd for C₂₆H₃₆N₄O₄, 469.28; found 469.5. Retention time (anal.HPLC: 2-50% MeCN/H₂O over 5 min)=3.94 min.

Example 18 Synthesis of1-isopropyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-3-(formyl-methyl-amino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

1-Isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(S)-2-hydroxy-3-methylaminopropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide(235mg, 0.36mmol) was dissolved in ethyl formate (5.0 mL). The mixturewas heated at 65° C. for 16 h, and then concentrated to dryness underreduced pressure. The product was taken up in acetic acid/water (1:1)(1.5 mL) and purified by HPLC chromatography. The purified fractionswere lyophilized to give the the title intermediate as thetrifluoroacetic acid salt (97.5 mg). (m/z): [M+H]⁺ calcd for C₂₅H₃₄N₄O₄,455.27; found 455.2. Retention time (anal. HPLC: 2-50% MeCN/H₂O over 5min)=3.87 min.

Example 19 Synthesis of5-bromo-1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

To a solution of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylicacid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide(500 mg, 0.99 mmol) dissolved in a mixture of acetonitrile (5 mL) andacetic acid (10 mL) was added bromine (0.30 mL, 5.9 mmol). The mixturewas stirred at ambient temperature for 12 h, and concentrated underreduced pressure, yielding a pale yellow oily residue. The residue wasdissolved in 20% acetonitrile in water (0.5% TFA) (5 mL), and purifiedby HPLC. The title compound was obtained as a major product and isolatedas a tifluoroacetic acid salt (200 mg) ¹H—NMR (CD₃OD): δ (ppm) 8.64 (s,1H), 7.97 (s, 1H), 7.72-7.70 (m, 2H), 4.18 (brm, 2H), 4.0 (br s, 1H),3.2-3.0 (m), 2.88 (s, 3H), 2.79 (s, 3H), 2.5-2.2 (m, 2H), 1.56 (d, 6H).(m/z): [M+H]⁺ calcd for C₂₅H₃₅BrN₄O₅S, 583.16; found 583.4. Retentiontime (anal. HPLC: 10-50% MeCN/H₂O over 6 min)=3.90 min.

Example 20 Alternative Synthesis of((R)-2-hydroxy-3-{3-[(1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-(1S,3R,5R)-8-azabicyclo[3.2.1]oct-8-yl}propyl)methylcarbamicacid tert-butyl ester a. Preparation ofmethyl-(S)-1-oxiranylmethylcarbamic acid tert-butyl ester

((S)-3-chloro-2-hydroxypropyl)-methylcarbamic acid tert-butyl ester(2.23 g, 10 mmol) was dissolved in THF (30 mL), and then an aqueoussodium hydroxide solution (0.48 g in 10 mL water) was added. The mixturewas stirred for 2 h. The mixture was then concentrated under reducedpressure to remove most of the THF, and the remaining aqueous solutionwas extracted into ethyl acetate, washing with water. The product wasdried over sodium sulfate, filtered and concentrated under reducedpressure to give the title intermediate as an oil (1.6 g). (m/z):[M+Na]⁺ calcd for C₉H₁₇NO₃, 210.10; found 210.1. ¹H—NMR (DMSO d6, 299.96MHz): δ (ppm) 1.32-1.42 (s, 9H), 2.69-2.73 (m, 1H), 2.75-2.85 (s, 3H),2.95-3.05 (br s, 1H), 3.10-3.15 (dm, 1H), 3.16-3.21 (d, 1H), 3.37-3.51(m, 2H).

b. Synthesis of((R)-2-hydroxy-3-{3-[(1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-(1S,3R,5R)-8-azabicyclo[3.2.1]oct-8-yl}propyl)methylcarbamicacid tert-butyl ester

Methyl-(S)-1-oxiranylmethylcarbamic acid tert-butyl ester (9.53 g, 51.1mmol), and 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-aza-bicyclo[3.2.1]oct-3-yl}amide (8.7 g, 25.6 mmol), weredissolved in methanol (100 mL). The mixture was heated at 80° C. for 2h, and then concentrated under reduced pressure. The resulting oil wastaken up in ethyl acetate and washed with saturated aqueous sodiumbicarbonate, followed by saturated aqueous sodium chloride. The organicswere dried over sodium sulfate, filtered, and concentrated under reducedpressure. The resulting oil was flash chromatographed (SiO₂, elutingwith 10% methanol/90% dichloromethane). Fractions containing productwere concentrated to give the title intermediate as a white solid (13.5g). (m/z): [M+H]⁺ calcd for C₂₉H₄₂N₄O₅, 527.33; found 527.6. Retentiontime (anal. HPLC: 2-50% MeCN/H₂O over 5 min)=4.75 min.

Example 21 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[2-hydroxy-3-(methanesulfonyl-ethylamino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

Following the procedure of Example 1 replacing methylamine withethylamine in step h, the title compound was prepared. (m/z): [M+H]⁺calcd for C₂₆H₃₈N₄O₅S, 519.28; found 519.2. Retention time (anal. HPLC:10-70% MeCN/H₂O over 6 min)=2.91 min.

Example 22 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonylamino)-propyl]-8-azabicyclo[3.2.1]oct-3-yl}amidea1. Preparation of (S)-2-oxiranylmethylisoindole-1,3-dione

To a cold solution of (S)-1-oxiranylmethanol (5 g, 67.5 mmol) andphthalimide (9.9 g, 67.3 mmol) in tetrahydrofuran (200 mL) in ice bathwas added triphenylphosphine (17.9 g, 68.2 mmol) and diethylazodicarboxylate (12.3 g, 70.6 mmol). The mixture was stirred at 0° C.for 2 h and at ambient temperature for 48 h. The mixture wasconcentrated under vacuum, and the oily residue was purified by flashsilica column chromatography, yielding the desired product (10.1 g) aspale yellow solid: R_(f)=0.51 in 1:1 EtOAc/hexane. ¹H—NMR (CDCl₃, 300MHz): δ (ppm) 7.76-7.64 (m, 2H), 7.63-7.60 (m, 2H), 3.9-3.8 (dd, 1H),3.70-3.65 (dd, 1H), 3.15 (m, 1H), 2.70-2.67 (dd, 1H), 2.58-2.55 (dd,1H).

b1. Synthesis of 1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylicacid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-methanesulfonylamino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

Following the procedure of Example 4, replacing racemic2-oxiranylmethyl-isoindole-1,3-dione with the chiral intermediate of theprevious step, the trifluoroacetate salt of the title compound wasprepared. (m/z): [M+H]⁺ calcd for C₂₄H₃₄N₄O₅S, 491.24; found 491.4.Retention time (anal. HPLC: 10-50% MeCN/H₂O over 6 min)=3.69 min. ¹H—NMR(CD₃OD, 300 MHz): δ (ppm) 8.67 (s, 1H), 7.74-7.73 (m, 3H), 7.7-7.6 (dt,1H), 7.3-7.2 (t, 1H), 4.2 (br s, 2H), 4.0 (br m, 2H), 3.2-2.9 (m, 4H),2.8 (s, 3H), 2.6-2.3 (br m, 6H), 2.2-2.1 (br m, 2H), 1.57-1.55 (d, 6H).

The title compound was also prepared by the following procedure.

a2. Preparation of N-((S)-oxiranylmethyl methanesulfonamide

To a cold solution of methanesulfonamide (10 g, 0.105 mol) in water (100mL) in an ice bath was added sodium hydroxide as pellets (8.4 g, 0.21mol) and then (S)-2-chloromethyloxirane (12.4 g, 0.158 mol). The mixturewas stirred at the same temperature for 2 h, and at room temperature for12 h and then concentrated hydrochloric acid (18 mL) was added. Theproduct was isolated by extracting the aqueous layer withdichloromethane (2×300 mL). The organic layer was dried over MgSO₄ andthen evaporated to dryness, yielding a colorless liquid (2.5 g), whichwas used directly in the next step.

b2. Synthesis of 1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylicacid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonylamino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

To a solution of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylicacid {(1S,3R,5R)-8-aza-bicyclo[3.2.1]oct-3-yl}amide (TFA salt; 4 g, 8.8mmol) in methanol (150 mL) was added N,N-diisopropylethylamine (1.7 mL,9.5 mmol), and the product of the previous step (2.5 g, 18.2 mmol). Themixture was stirred at 80° C. for 2 days. After being concentrated undervacuum, the residue was purified by preparative HPLC, yielding thetrifluoroacetate salt of the title compound (1.3 g). (m/z): [M+H]⁺ calcdfor C₂₄H₃₄N₄O₅S, 491.24; found 491.4. Retention time (anal. HPLC: 10-50%MeCN/H₂O over 6 min)=3.69 min.

Example 23 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[2-hydroxy-3-(1,1-dioxo-2-isothiazolidinyl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amidea. Preparation of 1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylicacid{(1S,3R,5R)-8-[2-hydroxy-3-(3-chloropropanesulfonylamino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

To a cold solution of1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[2-hydroxy-3-aminopropyl]-8-azabicyclo[3.2.1]oct-3-yl}amideTFA salt (0.125 g, 0.195 mmol) in dichloromethane (2 mL) in an ice bathwas added N,N-diisopropylethylamine (0.119 mL, 0.683 mmol) and3-chloropropanesulfonyl chloride (0.025 mL, 0.205 mmol). After stirringat 0° C. for 2 h, the mixture was stirred at room temperature overnight.It was diluted with dichloromethane (50 mL), and washed with brine andsaturated NaHCO₃ solution. After drying over MgSO₄, the organic solutionwas evaporated to dryness, yielding an oily residue. The crude productwas used directly in next step.

b. 1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylic acid{(1S,3R,5R)-8-[2-hydroxy-3-(1,1-dioxo-2-isothiazolidinylpropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

To a solution of 1-isopropyl-2-oxo-1,2-dihydroquinolinone-3-carboxylicacid{(1S,3R,5R)-8-[2-hydroxy-3-(3-chloropropanesulfonylamino)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide(100 mg, 0.18 mmol) in anhydrous DMF (3 mL) was added potassiumcarbonate (75 mg, 0.56 mmol). The reaction mixture was shaken at 85° C.for 12 h, and concentrated under vacuum. The residue was dissolved indichloromethane (50 mL), and washed with saturated NaHCO₃. After dryingover MgSO₄, the filtrate was evaporated to dryness, and the residue waspurified by preparative HPLC to yield the title compound. (m/z): [M+H]⁺calcd for C₂₆H₃₆N₄O₅S, 517.26; found 517.3.

Example 24 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amidea. Preparation of(1S,3R,5R)-3-[1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-8-azabicyclo[3.2.1]octane-8-carboxylicacid tert-butyl ester

In a 3 L flask, 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid(112.4 g, 0.486 mol, 1.1 eq) was suspended in toluene (1 L). The mixturewas heated to 85° C. and thionyl chloride (86.74 g, 0.729 mol) was addeddropwise over 70 min. The mixture was heated at 95° C. for 1.5 h withstirring and then allowed to cool to room temperature.

In a separate 12 L flask,(1S,3R,5R)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylic acidtert-butyl ester (100.0 g, 0.442 mol, 1 eq) was suspended in toluene (1L) and 3 M NaOH (4 eq) was added. The mixture was stirred at roomtemperature for 10 min and then cooled to about 5° C. The acid chloridesolution was added slowly with stirring over 40 min keeping the internaltemperature below 10° C. The mixture was stirred at 3-5° C. for 30 minand the layers were allowed to separate overnight. The toluene layer(˜2.5 L) was collected, concentrated to about half (˜1.2 L) by rotaryevaporation, and used directly in the next step.

b. Preparation of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylicacid {(1S,3R,5R)-8-azabicyclo[3.2.1]oct-3-yl}amide

To the toluene solution prepared in the previous step (˜1.2 L) was addedtrifluoroacetic acid (200 mL) over 20 min at 20° C. with stirring. Themixture was stirred at 20° C. for 2 h. Water (1.55 L) was added and themixture was stirred for 30 min at 20° C. After 30 min, the mixtureseparated into three layers. The bottom layer (˜350 mL), a viscous brownoil, contained the crude intermediate.

To a 12 L flask charged with MTBE (2.8 L), the crude brown oil was addedover 1 h at 1-2° C. with stirring. The suspension was stirred at thesame temperature for 1 h and then filtered. The filtrate was washed withMTBE (2×300 mL) and dried under vacuum at room temperature for 4 days toprovide the trifluoroacetate salt of the title intermediate (163.3 g) asa pale yellow powder.

c. Preparation of N-methyl-N-[(S)-2-oxiran-2-ylmethyl]methanesulfonamide

A 12 L flask was charged with water (1 L) followed by the addition NaOH(50% in water, 146.81 g, 1.835 mol). The beaker containing NaOH waswashed with water (2×500 mL) and the washings were added to the flask.The mixture was stirred at room temperature for 10 min and cooled to 8°C. (N-methyl)methanesulfonamide (200.2 g, 1.835 mol) in water (500 mL)was added over 5 min. The mixture was stirred for 1 h at ˜4° C. and(5)-2-chloromethyloxirane (339.6 g, 3.67 mol) was added. The mixture wasstirred for 20 h at 3-4° C. Dichloromethane (2 L) was added and themixture was stirred for 30 min at 5-10° C. The two layers were allowedto separate over 10 min and collected. The organic layer (˜2.5 L) wasadded back to the 12 L flask and washed with 1 M H₃PO₄ (800 mL) andbrine (800 mL). Dichloromethane was removed by rotary evaporation. Tothe crude product, toluene (400 mL) was added and removed by rotaryevaporation. After three additional cycles of the toluene process, thetitle intermediate was obtained (228.2 g) which was used without furtherpurification in the next step.

d. Synthesis of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amide

In a 3 L flask, 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-azabicyclo[3.2.1]oct-3-yl}amide trifluoroacetate (105.0 g,0.232 mol) was suspended in absolute ethanol (400 mL). To thissuspension, NaOH (50% in water, 0.243 mol. 1.05 eq) dissolved inabsolute ethanol (100 mL) was added at room temperature. The beakercontaining the NaOH was washed with ethanol (2×50 mL) and the washingswere added to the reaction mixture. After 30 min of stirring, a solutionof N-methyl-N-[(S)-2-oxiran-2-ylmethyl]methanesulfonamide (62.0 g, 1.5eq) in absolute ethanol (100 mL) was added. The mixture was refluxed for2 h, cooled to room temperature and seed crystals of the title compoundwere added. After about 5 min of stirring a white solid formed. Themixture was cooled to 3-5° C. and stirred for 2 h. The white solid wasfiltered and the wet cake was washed with cold absolute ethanol (3×50mL). The solid was dried under vacuum at 30° C. for 60 h to provide thetitle compound (93.8 g, water content by Karl Fischer method 2.03%). ¹HNMR (CDCl₃) δ ppm 10.52 (d, 1H), 8.83 (s, 1H), 7.75 (d, 2H), 7.64-7.60(m, 2H), 7.28-7.26 m, 1H), 4.33-4.26 (m, 2H), 3.78-3.75 (m, 1H),3.27-3.20 (m, 4H), 3.01 (s, 3H), 2.88 (s, 3H), 2.58-2.53 (m, 1H),2.30-1.81(m, 11H), 1.68 (d, 6H).

The seed crystals were obtained from a previous preparation of the titlecompound by the method of this example at smaller scale, in whichcrystallization occurred spontaneously.

Example 25 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amidehydrochloride

In a 1 L flask, 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amide(34.7 g, 0.069 mol) was suspended in absolute ethanol (210 mL).Concentrated HCl (1.1 eq) was added at room temperature with stirring.The mixture was stirred at reflux for 30 min and cooled to roomtemperature and stirred for 2 h. The solid was filtered and the wet cakewas washed with cold absolute ethanol (3×50 mL). The solid was driedunder vacuum at 30° C. for 48 h to provide the title compound (34.5 g,93.7% yield, water content by Karl Fischer method 0.13%).

Example 26 Synthesis of citric acid salt of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amide

1-Isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amide(0.1 g, 0.2 mmol) was suspended in ethanol (1 mL). To this suspensionwas added a 1M solution of citric acid in ethanol (0.072 mL, 0.072 mmol,0.33 eq). The mixture was briefly sonicated until clarity, capped, andthen allowed to sit overnight. The cap was then removed and the mixturewas allowed to evaporate under ambient conditions until solids wereobserved. The mixture was then recapped and allowed to sit for 72 h. Theresulting solid was filtered and washed with cold ethanol to give thetitle compound as a solid (74.3 mg).

Example 27 Synthesis of acid salts of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amide

Following the procedure of Example 26, the acid salts of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amidelisted below in Table III were prepared in solid form using theindicated equivalents of acid.

TABLE III Acid Salts Acid No. of equivalents of acid Product weight (mg)adipic 0.5 48.5 phosphoric 0.5 86.6 sulfuric 0.5 27.0 tartaric 0.5 66.3malic 0.5 25.3 hydrobromic 1 62.9

Example 28 Synthesis of methanesulfonic acid salt of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amide

To a solution of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylicacid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amide(0.1 g, 0.2 mmol) in 50% acetonitrile/water (1 mL) was added a 1Msolution of methanesulfonic acid in ethanol (0.2 mL, 0.2 mmol, 1 eq).The mixture was then frozen and lyophilized to dryness overnight. Theresulting solid was dissolved in isopropanol (1 mL) with gentle warmingand allowed to cool. The resulting solid was collected by filtration andwashed with cold isopropanol to give the title compound as a solid (90mg).

Example 29 Synthesis of acid salts of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amide

Following the procedure of Example 28, the acid salts of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(methanesulfonyl-methyl-amino)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amidelisted below in Table IV were prepared in solid form using the indicatedequivalents of acid.

TABLE IV Acid Salts Acid No. of equivalents of acid Product weight (mg)fumaric 1 107.2 benzoic 1 105.0 (R)-mandelic 1 96.1

Assay 1: Radioligand Binding Assay on 5-HT_(4(c)) Human Receptors a.Membrane Preparation 5-HT_(4(c))

HEK-293 (human embryonic kidney) cells stably-transfected with human5-HT_(4(c)) receptor cDNA (Bmax=˜6.0 pmol/mg protein, as determinedusing [³H]-GR113808 membrane radioligand binding assay) were grown inT-225 flasks in Dulbecco's Modified Eagles Medium (DMEM) containing4,500 mg/L D-glucose and pyridoxine hydrochloride (GIBCO-InvitrogenCorp., Carlsbad Calif.: Cat #11965) supplemented with 10% fetal bovineserum (FBS) (GIBCO-Invitrogen Corp.: Cat #10437), 2 mM L-glutamine and(100 units) penicillin-(100 μg) streptomycin/ml (GIBCO-Invitrogen Corp.:Cat #15140) in a 5% CO₂, humidified incubator at 37° C. Cells were grownunder continuous selection pressure by the addition of 800 μg/mLgeneticin (GIBCO-Invitrogen Corp.: Cat #10131) to the medium.

Cells were grown to roughly 60-80% confluency (<35 subculture passages).At 20-22 hours prior to harvesting, cells were washed twice and fed withserum-free DMEM. All steps of the membrane preparation were performed onice. The cell monolayer was lifted by gentle mechanical agitation andtrituration with a 25 mL pipette. Cells were collected by centrifugationat 1000 rpm (5 min).

For the membrane preparation, cell pellets were resuspended in ice-cold50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), pH7.4 (membrane preparation buffer) (40 mL/total cell yield from 30-40T225 flasks) and homogenized using a polytron disrupter (setting 19,2×10 s) on ice. The resultant homogenates were centrifuged at 1200 g for5 min at 4° C. The pellet was discarded and the supernatant centrifugedat 40,000 g (20 min). The pellet was washed once by resuspension withmembrane preparation buffer and centrifugation at 40,000 g (20 min). Thefinal pellet was resuspended in 50 mM HEPES, pH 7,4 (assay buffer)(equivalent 1 T225 flask/1 mL). Protein concentration of the membranesuspension was determined by the method of Bradford (Bradford, 1976).Membranes were stored frozen in aliquots at −80° C.

b. Radioligand Binding Assays

Radioligand binding assays were performed in 1.1 mL 96-deep wellpolypropylene assay plates (Axygen) in a total assay volume of 400 μLcontaining 2 μg membrane protein in 50 mM HEPES pH 7.4, containing0.025% bovine serum albumin (BSA). Saturation binding studies fordetermination of K_(d) values of the radioligand were performed using[³H]-GR113808 (Amersham Inc., Bucks, UK: Cat #TRK944; specific activity˜82 Ci/mmol) at 8-12 different concentrations ranging from 0.001 nM-5.0nM. Displacement assays for determination of pK_(i) values of compoundswere performed with [³H]-GR113808 at 0.15 nM and eleven differentconcentrations of compound ranging from 10 pM-100 μM.

Test compounds were received as 10 mM stock solutions in DMSO anddiluted to 400 μM into 50 mM HEPES pH 7.4 at 25° C., containing 0.1%BSA, and serial dilutions (1:5) then made in the same buffer.Non-specific binding was determined in the presence of 1 μM unlabeledGR113808. Assays were incubated for 60 min at room temperature, and thenthe binding reactions were terminated by rapid filtration over 96-wellGF/B glass fiber filter plates (Packard BioScience Co., Meriden, Conn.)presoaked in 0.3% polyethyleneimine. Filter plates were washed threetimes with filtration buffer (ice-cold 50 mM HEPES, pH7.4) to removeunbound radioactivity. Plates were dried, 35 μL Microscint-20 liquidscintillation fluid (Packard BioScience Co., Meriden, Conn.) was addedto each well and plates were counted in a Packard Topcount liquidscintillation counter (Packard BioScience Co., Meriden, Conn.).

Binding data were analyzed by nonlinear regression analysis with theGraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) using the 3-parameter model for one-site competition. The BOTTOM(curve minimum) was fixed to the value for nonspecific binding, asdetermined in the presence of 1 μM GR113808. K_(i) values for testcompounds were calculated, in Prism, from the best-fit IC₅₀ values, andthe K_(d) value of the radioligand, using the Cheng-Prusoff equation(Cheng and Prusoff, Biochemical Pharmacology, 1973, 22, 3099-108):K_(i)=IC₅₀/(1+[L]/K_(d)) where [L]=concentration [³H]-GR113808. Resultsare expressed as the negative decadic logarithm of the K_(i) values,pK_(i).

Test compounds having a higher pK_(i) value in this assay have a higherbinding affinity for the 5-HT₄ receptor. The compounds of the inventionwhich were tested in this assay had a pK_(i) value ranging from about6.3 to about 9.0, typically ranging from about 6.5 to about 8.5.

Assay 2: Radioligand Binding Assay on 5-HT_(3A) Human Receptors

Determination of Receptor Subtype Selectivity

a. Membrane Preparation 5-HT_(3A)

HEK-293 (human embryonic kidney) cells stably-transfected with human5-HT_(3A) receptor cDNA were obtained from Dr. Michael Bruess(University of Bonn, GDR) (Bmax=˜9.0 pmol/mg protein, as determinedusing [³H]-GR65630 membrane radioligand binding assay). Cells were grownin T-225 flasks or cell factories in 50% Dulbecco's Modified EaglesMedium (DMEM) (GIBCO-Invitrogen Corp., Carlsbad, Calif.: Cat #11965) and50% Ham's F12 (GIBCO-Invitrogen Corp.: Cat #11765) supplemented with 10%heat inactivated fetal bovine serum (FBS) (Hyclone, Logan, Utah: Cat#SH30070.03) and (50 units) penicillin-(50 μg) streptomycin/ml(GIBCO-Invitrogen Corp.: Cat #15140) in a 5% CO₂, humidified incubatorat 37° C.

Cells were grown to roughly 70-80% confluency (<35 subculture passages).All steps of the membrane preparation were performed on ice. To harvestthe cells, the media was aspirated and cells were rinsed with Ca²⁺,Mg²⁺-free Dulbecco's phosphate buffered saline (dPBS). The cellmonolayer was lifted by gentle mechanical agitation. Cells werecollected by centrifugation at 1000 rpm (5 min). Subsequent steps of themembrane preparation followed the protocol described above for themembranes expressing 5-HT_(4(c)) receptors.

b. Radioligand Binding Assays

Radioligand binding assays were performed in 96-well polypropylene assayplates in a total assay volume of 200 μL containing 1.5-2 μg membraneprotein in 50 mM HEPES pH 7.4, containing 0.025% BSA assay buffer.Saturation binding studies for determination of K_(d) values of theradioligand were performed using [³H]-GR65630 (PerkinElmer Life SciencesInc., Boston, Mass.: Cat #NET1011, specific activity 85 Ci/mmol) attwelve different concentrations ranging from 0.005 nM to 20 nM.Displacement assays for determination of pK_(i) values of compounds wereperformed with [³H]-GR65630 at 0.50 nM and eleven differentconcentrations of compound ranging from 10 pM to 100 μM. Compounds werereceived as 10 mM stock solutions in DMSO (see section 3.1), diluted to400 μM into 50 nM HEPES pH 7.4 at 25° C., containing 0.1% BSA, andserial (1:5) dilutions then made in the same buffer. Non-specificbinding was determined in the presence of 10 μM unlabeled MDL72222.Assays were incubated for 60 min at room temperature, then the bindingreactions were terminated by rapid filtration over 96-well GF/B glassfiber filter plates (Packard BioScience Co., Meriden, Conn.) presoakedin 0.3% polyethyleneimine. Filter plates were washed three times withfiltration buffer (ice-cold 50 mM HEPES, pH7.4) to remove unboundradioactivity. Plates were dried, 35 μL Microscint-20 liquidscintillation fluid (Packard BioScience Co., Meriden, Conn.) was addedto each well and plates were counted in a Packard Topcount liquidscintillation counter (Packard BioScience Co., Meriden, Conn.).

Binding data were analyzed using the non-linear regression proceduredescribed above to determine K_(i) values. The BOTTOM (curve minimum)was fixed to the value for nonspecific binding, as determined in thepresence of 10 μM MDL72222. The quantity [L] in the Cheng-Prusoffequation was defined as the concentration [³ H]-GR65630.

Selectivity for the 5-HT₄ receptor subtype with respect to the 5-HT₃receptor subtype was calculated as the ratioK_(i)(5-HT_(3A))/K_(i)(5-HT_(4(c))). The compounds of the inventionwhich were tested in this assay had a 5-HT₄/5-HT₃ receptor subtypeselectivity ranging from about 50 to about 8000, typically ranging fromabout 100 to about 4000.

Assay 3: Whole-cell cAMP Accumulation Flashplate Assay with HEK-293Cells Expressing Human 5-HT_(4(c)) Receptors

In this assay, the functional potency of a test compound was determinedby measuring the amount of cyclic AMP produced when HEK-293 cellsexpressing 5-HT₄ receptors were contacted with different concentrationsof test compound.

a. Cell Culture

HEK-293 (human embryonic kidney) cells stably-transfected with clonedhuman 5-HT_(4(c)) receptor cDNA were prepared expressing the receptor attwo different densities: (1) at a density of about 0.5-0.6 pmol/mgprotein, as determined using a [³H]-GR113808 membrane radioligandbinding assay, and (2) at a density of about 6.0 pmol/mg protein. Thecells were grown in T-225 flasks in Dulbecco's Modified Eagles Medium(DMEM) containing 4,500 mg/L D-glucose (GIBCO-Invitrogen Corp.: Cat#11965) supplemented with 10% fetal bovine serum (FBS) (GIBCO-InvitrogenCorp.: Cat #10437) and (100 units) penicillin-(100 μg) streptomycin/ml(GIBCO-Invitrogen Corp.: Cat #15140) in a 5% CO₂, humidified incubatorat 37° C. Cells were grown under continuous selection pressure by theaddition of geneticin (800 μg/mL: GIBCO-Invitrogen Corp.: Cat #10131) tothe medium.

b. Cell Preparation

Cells were grown to roughly 60-80% confluency. Twenty to twenty-twohours prior to assay, cells were washed twice, and fed, with serum-freeDMEM containing 4,500 mg/L D-glucose (GIBCO-Invitrogen Corp.: Cat#11965). To harvest the cells, the media was aspirated and 10 mL Versene(GIBCO-Invitrogen Corp.: Cat #15040) was added to each T-225 flask.Cells were incubated for 5 min at RT and then dislodged from the flaskby mechanical agitation. The cell suspension was transferred to acentrifuge tube containing an equal volume of pre-warmed (37° C.) dPBSand centrifuged for 5 min at 1000 rpm. The supernatant was discarded andthe pellet was re-suspended in pre-warmed (37° C.) stimulation buffer(10 mL equivalent per 2-3 T-225 flasks). This time was noted and markedas time zero. The cells were counted with a Coulter counter (count above8 μm, flask yield was 1-2×10⁷ cells/flask). Cells were resuspended at aconcentration of 5×10⁵ cells/ml in pre-warmed (37° C.) stimulationbuffer (as provided in the flashplate kit) and preincubated at 37° C.for 10 min.

cAMP assays were performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with ¹²⁵I-cAMP(SMP004B, PerkinElmer Life Sciences Inc., Boston, Mass.), according tothe manufacturer's instructions.

Cells were grown and prepared as described above. Final cellconcentrations in the assay were 25×10³ cells/well and the final assayvolume was 100 μL. Test compounds were received as 10 mM stock solutionsin DMSO, diluted to 400 μM into 50 mM HEPES pH 7.4 at 25° C., containing0.1% BSA, and serial (1:5) dilutions then made in the same buffer.Cyclic AMP accumulation assays were performed with 11 differentconcentrations of compound ranging from 10 pM to 100 μM (final assayconcentrations). A 5-HT concentration-response curve (10 pM to 100 μM)was included on every plate. The cells were incubated, with shaking, at37° C. for 15 min and the reaction terminated by addition of 100 μl ofice-cold detection buffer (as provided in the flashplate kit) to eachwell. The plates were sealed and incubated at 4° C. overnight. Boundradioactivity was quantified by scintillation proximity spectroscopyusing the Topcount (Packard BioScience Co., Meriden, Conn.).

The amount of cAMP produced per mL of reaction was extrapolated from thecAMP standard curve, according to the instructions provided in themanufacturer's user manual. Data were analyzed by nonlinear regressionanalysis with the GraphPad Prism Software package using the 3-parametersigmoidal dose-response model (slope constrained to unity). Potency dataare reported as pEC₅₀ values, the negative decadic logarithm of the EC₅₀value, where EC₅₀ is the effective concentration for a 50% maximalresponse.

Test compounds exhibiting a higher pEC₅₀ value in this assay have ahigher potency for agonizing the 5-HT₄ receptor. The compounds of theinvention which were tested in this assay, for example, in the cell line(1) having a density of about 0.5-0.6 pmol/mg protein, had a pEC₅₀ valueranging from about 7.0 to about 9.0, typically ranging from about 7.5 toabout 8.5.

Assay 4: In vitro Voltage Clamp Assay of Inhibition of Potassium IonCurrent in Whole Cells Expressing the hERG Cardiac Potassium Channel

CHO-K1 cells stably transfected with hERG cDNA were obtained from GailRobertson at the University of Wisconsin. Cells were held in cryogenicstorage until needed. Cells were expanded and passaged in Dulbecco'sModified Eagles Medium/F12 supplemented with 10% fetal bovine serum and200 μg/mL geneticin. Cells were seeded onto poly-D-lysine (100 μg/mL)coated glass coverslips, in 35 mm² dishes (containing 2 mL medium) at adensity that enabled isolated cells to be selected for whole cellvoltage-clamp studies. The dishes were maintained in a humidified, 5%CO₂ environment at 37° C.

Extracellular solution was prepared at least every 7 days and stored at4° C. when not in use. The extracellular solution contained (mM): NaCl(137), KCl (4), CaCl₂ (1.8), MgCl₂ (1), Glucose (10),4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) (10), pH 7.4with NaOH. The extracellular solution, in the absence or presence oftest compound, was contained in reservoirs, from which it flowed intothe recording chamber at approximately 0.5 mL/min. The intracellularsolution was prepared, aliquoted and stored at −20° C. until the day ofuse. The intracellular solution contained (mM): KCl (130), MgCl₂ (1),ethylene glycol-bis(beta-aminoethyl ether) N,N,N′,N′-tetra acetic acidsalt (EGTA) (5), MgATP (5),4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) (10), pH 7.2with KOH. All experiments were performed at room temperature (20-22°C.).

The coverslips on which the cells were seeded were transferred to arecording chamber and perfused continuously. Gigaohm seals were formedbetween the cell and the patch electrode. Once a stable patch wasachieved, recording commenced in the voltage clamp mode, with theinitial holding potential at −80 mV. After a stable whole-cell currentwas achieved, the cells were exposed to test compound. The standardvoltage protocol was: step from the holding potential of −80 mV to +20mV for 4.8 sec, repolarize to −50 mV for 5 sec and then return to theoriginal holding potential (−80 mV). This voltage protocol was run onceevery 15 sec (0.067 Hz). Peak current amplitudes during therepolarization phase were determined using pClamp software. Testcompounds at a concentration of 3 μM were perfused over the cells for 5minutes, followed by a 5-minute washout period in the absence ofcompound. Finally a positive control (cisapride, 20 nM) was added to theperfusate to test the function of the cell. The step from −80 mV to +20mV activates the hERG channel, resulting in an outward current. The stepback to −50 mV results in an outward tail current, as the channelrecovers from inactivation and deactivates.

Peak current amplitudes during the repolarization phase were determinedusing pCLAMP software. The control and test article data were exportedto Origin® (OriginLab Corp., Northampton Mass.) where the individualcurrent amplitudes were normalized to the initial current amplitude inthe absence of compound. The normalized current means and standarderrors for each condition were calculated and plotted versus the timecourse of the experiment.

Comparisons were made between the observed K⁺ current inhibitions afterthe five-minute exposure to either the test article or vehicle control(usually 0.3% DMSO). Statistical comparisons between experimental groupswere performed using a two-population, independent t-test (MicrocalOrigin v. 6.0). Differences were considered significant at p <0.05.

The smaller the percentage inhibition of the potassium ion current inthis assay, the smaller the potential for test compounds to change thepattern of cardiac repolarization when used as therapeutic agents. Thecompounds of the invention which were tested in this assay at aconcentration of 3 μM exhibited an inhibition of the potassium ioncurrent of less than about 20%, typically, less than about 15%.

Assay 5: In vitro Model of Oral Bioavailability: Caco-2 Permeation Assay

The Caco-2 permeation assay was performed to model the ability of testcompounds to pass through the intestine and get into the blood streamafter oral administration. The rate at which test compounds in solutionpermeate a cell monolayer designed to mimic the tight junction of humansmall intestinal monolayers was determined.

Caco-2 (colon, adenocarcinoma; human) cells were obtained from ATCC(American Type Culture Collection; Rockville, Md.). For the permeationstudy, cells were seeded at a density of 63,000 cells/cm² on pre-wettedtranswells polycarbonate filters (Costar; Cambridge, Mass.). A cellmonolayer was formed after 21 days in culture. Following cell culture inthe transwell plate, the membrane containing the cell monolayer wasdetached from the transwell plate and inserted into the diffusionchamber (Costar; Cambridge, Mass.). The diffusion chamber was insertedinto the heating block which was equipped with circulating external,thermostatically regulated 37° C. water for temperature control. The airmanifold delivered 95% O₂/5% CO₂ to each half of a diffusion chamber andcreated a laminar flow pattern across the cell monolayer, which waseffective in reducing the unstirred boundary layer.

The permeation study was performed with test compound concentrations at100 μM and with ¹⁴C-mannitol to monitor the integrity of the monolayer.All experiments were conducted at 37° C. for 60 min. Samples were takenat 0, 30 and 60 min from both the donor and receiver sides of thechamber. Samples were analyzed by HPLC or liquid scintillation countingfor test compound and mannitol concentrations. The permeationcoefficient (K_(p)) in cm/sec was calculated.

In this assay, a K_(p) value greater than about 10×10⁻⁶ cm/sec isconsidered indicative of favorable bioavailability. The compounds of theinvention that were tested in this assay exhibited K_(p) values ofbetween about 10×10⁻⁶ cm/sec and about 50×10⁻⁶ cm/sec, typically betweenabout 20×10⁻⁶ cm/sec and about 40×10⁻⁶ cm/sec.

Assay 6: Pharmacokinetic Study in the Rat

Aqueous solution formulations of test compounds were prepared in 0.1%lactic acid at a pH of between about 5 and about 6. Male Sprague-Dawleyrats (CD strain, Charles River Laboratories, Wilmington, Mass.) weredosed with test compounds via intravenous administration (IV) at a doseof 2.5 mg/kg or by oral gavage (PO) at a dose of 5 mg/kg. The dosingvolume was 1 mL/kg for IV and 2 mL/kg for PO administration. Serialblood samples were collected from animals pre-dose, and at 2 (IV only),5, 15, and 30 min, and at 1, 2, 4, 8, and 24 hours post-dose.Concentrations of test compounds in blood plasma were determined byliquid chromatography-mass spectrometry analysis (LC-MS/MS) (DS SCIEX,API 4000, Applied Biosystems, Foster City, Calif.) with a lower limit ofquantitation of 1 ng/mL.

Standard pharmacokinetic parameters were assessed by non-compartmentalanalysis (Model 201 for IV and Model 200 for PO) using WinNonlin(Version 4.0.1, Pharsight, Mountain View, Calif.). The maximum in thecurve of test compound concentration in blood plasma vs. time is denotedC_(max). The area under the concentration vs. time curve from the timeof dosing to the last measurable concentration (AUC(0-t)) was calculatedby the linear trapezoidal rule. Oral bioavailability (F(%)), i.e. thedose-normalized ratio of AUC(0-t) for PO administration to AUC(0-t) forIV administration, was calculated as:F(%)=AUC_(PO)/AUC_(IV)×Dose_(IV)/Dose_(PO)×100%

Test compounds which exhibit larger values of the parameters C_(max),AUC(0-t), and F(%) in this assay are expected to have greaterbioavailability when administered orally. The compounds of the inventionthat were tested in this assay had C_(max) values typically ranging fromabout 0.1 to about 0.25 μg/mL and AUC(0-t) values typically ranging fromabout 0.4 to about 0.9 μg·hr/mL. By way of example, the compound ofExample 1 had a C_(max) value of 0.17 μg/mL, an AUC(0-t) value of 0.66μg·hr/mL and oral bioavailability (F(%)) in the rat model of about 35%.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. Additionally, all publications, patents, andpatent documents cited hereinabove are incorporated by reference hereinin full, as though individually incorporated by reference.

1. A capsule comprising a pharmaceutically acceptable carrier and acompound of the formula

or a pharmaceutically-acceptable salt thereof.
 2. The capsule of claim 1wherein the pharmaceutically acceptable carrier comprises one or moreingredients selected from fillers and lubricants.
 3. The capsule ofclaim 1 wherein the pharmaceutically acceptable carrier comprises one ormore ingredients selected from starches, microcrystalline cellulose,lactose, sucrose, glucose, mannitol, silicic acid, talc, calciumstearate, magnesium stearate, solid polyethylene glycols, and sodiumlauryl sulfate.
 4. The capsule of claim 1 wherein the pharmaceuticallyacceptable carrier comprises one or more ingredients selected fromstarches, microcrystalline cellulose and magnesium stearate.
 5. Acapsule comprising a hydrochloride salt of a compound of the formula

and a pharmaceutically acceptable carrier.
 6. The capsule of claim 5wherein the pharmaceutically acceptable carrier comprises one or moreingredients selected from fillers and lubricants.
 7. The capsule ofclaim 5 wherein the pharmaceutically acceptable carrier comprises one ormore ingredients selected from starches, microcrystalline cellulose,lactose, sucrose, glucose, mannitol, silicic acid, talc, calciumstearate, magnesium stearate, solid polyethylene glycols, and sodiumlauryl sulfate.
 8. The capsule of claim 5 wherein the pharmaceuticallyacceptable carrier comprises one or more ingredients selected fromstarches, microcrystalline cellulose and magnesium stearate.
 9. A methodof treating a disorder of reduced motility of the gastrointestinal tractin a mammal, the method comprising administering to the mammal a capsuleof claim
 1. 10. The method of claim 9, wherein the disorder of reducedmotility is chronic constipation, constipation-predominant irritablebowel syndrome, diabetic gastroparesis, drug-induced delayed transit, orfunctional dyspepsia.
 11. A method of treating chronic constipation orconstipation-predominant irritable bowel syndrome in a mammal, themethod comprising administering to the mammal a capsule of claim
 1. 12.A method of treating a disorder of reduced motility of thegastrointestinal tract in a mammal, the method comprising administeringto the mammal a capsule of claim
 5. 13. The method of claim 12, whereinthe disorder of reduced motility is chronic constipation,constipation-predominant irritable bowel syndrome, diabeticgastroparesis, drug-induced delayed transit, or functional dyspepsia.14. A method of treating chronic constipation orconstipation-predominant irritable bowel syndrome in a mammal, themethod comprising administering to the mammal a capsule of claim
 5. 15.The capsule of claim 1 wherein the pharmaceutically acceptable carriercomprises one or more ingredients selected from lactose and magnesiumstearate.
 16. The capsule of claim 1 wherein the pharmaceuticallyacceptable carrier comprises one or more ingredients selected frommicrocrystalline cellulose and magnesium stearate.
 17. The capsule ofclaim 5 wherein the pharmaceutically acceptable carrier comprises one ormore ingredients selected from lactose and magnesium stearate.
 18. Thecapsule of claim 5 wherein the pharmaceutically acceptable carriercomprises one or more ingredients selected from microcrystallinecellulose and magnesium stearate.